Application of click chemistry for signal amplification in IHC and ISH assays

Abstract

Applicants have developed amplification systems and methods for IHC and ISH staining that utilize "click chemistry" to covalently bind reporter molecules to tissue.

Description

[0001] Statement of Industrial Applicability

[0002] The present disclosure has industrial applicability in the fields of chemistry and diagnostics.

[0003] Public background

[0004] Immunohistochemistry (IHC) refers to the process of detecting, locating and / or quantifying antigens (such as proteins) in biological samples using antibodies specific for specific antigens. IHC provides the substantial advantage of accurately determining the position of a specific protein in a tissue sample. It is also an effective way to inspect the tissue itself. In situ hybridization (ISH) refers to the process of detecting, locating and quantifying nucleic acids. Both IHC and ISH can be performed on various biological samples (such as tissues (e.g., fresh frozen, formalin fixed, paraffin embedded) and cytological samples). Whether the target is nucleic acid or antigen, the identification of the target can be detected using various labels (e.g., color development, fluorescence, luminescence, radiation). In order to robustly detect, locate and quantify targets in a clinical setting, amplification recognition events are desirable because the ability to confidently detect low abundance cell markers becomes increasingly important for diagnostic purposes. For example, in response to a single antigen detection event, hundreds or thousands of marker molecules are deposited at the site of the marker, enhancing the ability to detect the recognition event by amplification.

[0005] Adverse events are often accompanied by amplification, such as the non-specific signal that manifests as the background signal of increase.The background signal of increase interferes with clinical analysis by blurring the faint signal that may be relevant to low but clinically significant expression.Therefore, although the amplification of recognition event is desirable, the amplification method that does not increase background signal is highly desirable.A kind of this method is tyramide signal amplification (TSA), which is also referred to as the reporter sub-deposition (CARD) of catalysis.U.S. Patent number 5,583,001 discloses the method for using analyte-dependent enzyme activation system to detect and / or quantitative analyte, and the system relies on the reporter sub-deposition of catalysis to amplify detectable label signal.By making the phenol molecule of labeling and enzyme reaction, strengthen the catalysis of enzyme in CARD or TSA method.Utilize the modern method of TSA to effectively increase the signal that obtains from IHC and ISH determination, do not produce significant background signal amplification simultaneously (for the disclosure relevant to tyramide amplification reagent, see, for example, U.S. application publication number 2012 / 0171668, which is incorporated by reference with its entirety). Reagents for these amplification methods are being applied to clinically important targets to provide robust diagnostic capabilities that were previously unavailable (OPTIVIEW® Amplification Kit, Ventana Medical Systems, Tucson AZ, catalog number 760-099).

[0006] TSA utilizes the reaction between horseradish peroxidase (HRP) and junket amide.In the presence of H o , junket amide is converted into highly reactive and short-lived free radical intermediates, which preferentially react with the amino acid residue that is rich in electronics on the albumen.Then, covalently bound detectable markers can be detected by various color development techniques and / or by fluorescence microscopy.In the solid phase immunoassay (such as IHC and ISH) that attaches great importance to space and morphological background, the short-lived free radical intermediate causes the covalent binding of junket amides to the protein on the tissue that the site is closely approached, provides discrete and specific signal.

[0007] Co-pending application PCT / EP2015 / 0533556, entitled "Quinone Methide Analog Signal Amplification," with an international filing date of February 20, 2015, describes an alternative technique ("QMSA") that, like TSA, can be used to increase signal amplification without increasing background signal. In fact, PCT / EP2015 / 0533556 describes novel quinone methide analog precursors and methods for detecting one or more targets in biological samples using quinone methide analog precursors. Among them, the detection method is described as comprising the steps of contacting the sample with a detection probe and then contacting the sample with a labeled conjugate comprising an enzyme. The enzyme interacts with the quinone methide analog precursor comprising a detectable label to form a reactive quinone methide analog that binds to the biological sample close to or directly on the target. The detectable label is then detected.

[0008] "Click chemistry" is a chemical concept independently defined by the groups of Sharpless and Meldal that describes a chemical method that can be tuned to rapidly and reliably generate substances by linking small units together. "Click chemistry" has been applied to a collection of reliable and self-directed organic reactions (Kolb, HC; Finn, MG; Sharpless, KBAngew. Chem. Int. Ed. 2001, 40, 2004-2021). For example, the identification of copper-catalyzed azide-alkyne [3+2] cycloadditions as highly reliable molecular ligations in water (Rostovtsev, VV; et al. Angew. Chem. Int. ed. 2002, 41, 2596-2599) has been used to enhance the study 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). In addition, it has also appeared in organic synthesis (Lee, LV et al., J. Am. Chem. Soc. 2003, 125,9588-9589), drug discovery (Kolb, HC; Sharpless, KB Drug 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 2004, ASAP, in press; Lummerstorfer, T.; Hoffmann, HJ Phys. Chem. B 2004, in press).

[0009] In general, click chemistry promotes reactions that have a wide range of modular applications, have high chemical yields, generate harmless byproducts, are chemically specific, require simple reaction conditions, use readily available starting materials and reagents, are solvent-free or use benign solvents (such as water), result in easy product isolation, have a large thermodynamic driving force to favor reactions with a single reaction product, and have high atom economy. Although certain general criteria may be subjective in nature, not all criteria need to be met.

[0010] Public Description

[0011] The applicant has developed an amplification system and method for IHC and ISH staining that utilizes "click chemistry" to covalently bind reporter molecules to tissues. As will be further described herein, the amplification method of the present disclosure allows the reporter moiety to be separated from the QMSA or TSA assay conditions and thus provides advantages over QMSA and TSA protocols.

[0012] In one aspect of the present disclosure is a conjugate of formula (IIa):

[0013]

[0014] in,

[0015] A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine and hydroxylamine;

[0016] A "linker" is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N or S;

[0017] R 1 is a group selected from a phosphate group, an amide group, a nitro group, a urea group, a sulfate group, a methyl group, an ester group, a β-lactam group or a sugar group;

[0018] R 2 It is a halogen;

[0019] R 3 、R 5 and R 6 independently selected from hydrogen or an aliphatic group having 1 to 4 carbon atoms;

[0020] R 4 is hydrogen, an aliphatic group having 1 to 4 carbon atoms or a group -CH(R 2 )-R 7 -[Connector]-A; and

[0021] R 7 Selected from -(CH2)w NH-, -O(CH2) w NH-, -N(H)C(O)(CH2) w NH-、-C(O)N(H)(CH2) w NH-, -(CH2) w O-, -O(CH2) w O-, -O(CH2CH2O) w -、-N(H)C(O)(CH2) w O-, -C(O)N(H)(CH2) w O-, -C(O)N(H)(CH2CH2O) w -、-(CH2) w S-, -O(CH2) w S-, -N(H)C(O)(CH2) w S-、-C(O)N(H)(CH2) w S-, -(CH2) w NH-, -C(O)N(H)(CH2CH2O) w CH2CH2NH, -C(O)(CH2CH2O) w CH2CH2NH-, -C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2) w NH- or -N(H)(CH2) w NH-, wherein w is an integer ranging from 1 to 12.

[0022] In some embodiments, R 6 、R 5 、R 4 and R 3 In some embodiments, R 1 In some embodiments, R 2 In some embodiments, R 1 is phosphoric acid; R 2 is fluorine; and R 6 、R 5 、R 4 and R 3 Each is hydrogen.

[0023] In some embodiments, the "linker" has formula (Ia):

[0024]

[0025] in

[0026] d and e are each independently an integer ranging from 2 to 20;

[0027] t and u are independently 0 or 1;

[0028] Q is a bond, O, S or N (R c )(R d );

[0029] R a and R b are independently H, C1-C4 alkyl, F, Cl or -N(R c )(R d );

[0030] R c and R d are independently CH3 or H; and

[0031] X and Y are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally having one or more O, N or S heteroatoms.

[0032] In some embodiments, R a and R b In some embodiments, Q is oxygen. In some embodiments, R 7 is -C(O)N(H)(CH2) w NH-. In some embodiments, R 1 is phosphoric acid and R 7 is -C(O)N(H)(CH2) w NH-, and w ranges from 2 to 10. In some embodiments, is fluoro; and R 6 、R 5 、R 4 and R 3 Each is hydrogen. In some embodiments, the "linker" comprises a PEG group.

[0033] In another aspect of the present disclosure are conjugates of formula (IId):

[0034]

[0035] in

[0036] A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine and hydroxylamine;

[0037] A "linker" is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and

[0038] w ranges from 1 to 12.

[0039] In some embodiments, the "linker" has formula (Ia):

[0040]

[0041] in

[0042] d and e are each independently an integer ranging from 2 to 20;

[0043] t and u are independently 0 or 1;

[0044] Q is a bond, O, S or N (R c )(R d );

[0045] R a and R b are independently H, C1-C4 alkyl, F, Cl or N(R c )(R d );

[0046] R c and R d are independently CH3 or H; and

[0047] X and Y are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally having one or more O, N or S heteroatoms.

[0048] In some embodiments, w ranges from 1 to 8; and wherein R a and R b Each is hydrogen. In some embodiments, w ranges from 2 to 8, and wherein Q is oxygen. In some embodiments, d and e are independently integers ranging from 2 to 10. In some embodiments, A is dibenzocyclooctyne. In some embodiments, w ranges from 2 to 6, and wherein the linker comprises a PEG group. In some embodiments, A is trans-cyclooctene. In some embodiments, ranges from 2 to 6, and wherein the linker comprises a PEG group. In some embodiments, A is azide. In some embodiments, w ranges from 2 to 6, and wherein the linker comprises a PEG group. In some embodiments, A is tetrazine. In some embodiments, w ranges from 2 to 6, and wherein the linker comprises a PEG group.

[0049] In another aspect of the present disclosure are conjugates of formula (III):

[0050]

[0051] in

[0052] M is derived from propionic acid, cinnamic acid or a compound of formula (IIIa) having the following structure:

[0053]

[0054] wherein each R group is independently selected from hydrogen or a lower alkyl group having 1 to 4 carbon atoms (which may be straight or branched);

[0055] A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; and

[0056] A "linker" is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N or S;

[0057] with the proviso that when each R is hydrogen, A is selected from azide, thiol, 1,3-nitrone, hydrazine, or hydroxylamine.

[0058] In some embodiments, the "linker" has formula (Ia):

[0059]

[0060] in

[0061] d and e are each independently an integer ranging from 2 to 20;

[0062] t and u are independently 0 or 1;

[0063] Q is a bond, O, S or N (R c )(R d );

[0064] R a and R b are independently H, C1-C4 alkyl, F, Cl or N(R c )(R d );

[0065] R c and R d are independently CH3 or H; and

[0066] X and Y are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally having one or more O, N or S heteroatoms.

[0067] In some embodiments, R a and R bIn some embodiments, Q is oxygen. In some embodiments, R a and R b Each is hydrogen, Q is oxygen, and e ranges from 2 to 10.

[0068] In another aspect of the present disclosure are conjugates of formula (Id):

[0069]

[0070] in

[0071] A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine and hydroxylamine;

[0072] A "linker" is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and

[0073] The "tissue reactive pro-moiety" is derived from a compound selected from the group consisting of:

[0074]

[0075]

[0076]

[0077] In some embodiments, the "linker" has formula (Ia):

[0078]

[0079] in

[0080] d and e are each independently an integer ranging from 2 to 20;

[0081] t and u are independently 0 or 1;

[0082] Q is a bond, O, S or N (R c )(R d );

[0083] R a and R b are independently H, C1-C4 alkyl, F, Cl or N(R c )(R d );

[0084] R c and R d are independently CH3 or H; and

[0085] X and Y are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally having one or more O, N or S heteroatoms.

[0086] In some embodiments, R a and R b In some embodiments, Q is oxygen. In some embodiments, R a and R b Each is hydrogen, Q is oxygen, and e ranges from 2 to 10.

[0087] In another aspect of the present disclosure are conjugates of formula (IV):

[0088]

[0089] in

[0090] A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine and hydroxylamine;

[0091] A "linker" is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and

[0092] Z is selected from the group consisting of a chromophore, a fluorophore, an enzyme, a hapten, and a chelator.

[0093] In some embodiments, Z is a chromophore selected from tetramethylrhodamine, cyanine 5, and Dabsyl. In some embodiments, Z is selected from:

[0094]

[0095]

[0096]

[0097] In some embodiments, the conjugate has the structure of Formula (IVa):

[0098] .

[0099] In some embodiments, the conjugate has the structure of Formula (IVb):

[0100] .

[0101] In some embodiments, the conjugate has the structure of Formula (IVc):

[0102] .

[0103] In some embodiments, the conjugate has the structure of Formula (IVd):

[0104] .

[0105] In some embodiments, the conjugate is:

[0106] .

[0107] In some embodiments, the conjugate is:

[0108] .

[0109] In some embodiments, the conjugate is:

[0110] .

[0111] In some embodiments, the conjugate is:

[0112] .

[0113] In some embodiments, the conjugate is:

[0114] .

[0115] In some embodiments, the conjugate is:

[0116] .

[0117] In some embodiments, the conjugate is:

[0118] .

[0119] In some embodiments, the conjugate is:

[0120] .

[0121] Another aspect of the present disclosure is a method for detecting a first target in a biological sample, comprising: contacting the biological sample with a first detection probe specific for the first target to form a first detection probe-target complex; contacting the biological sample with a first labeled conjugate specific for the first detection probe, the first labeled conjugate comprising a first enzyme, such that the first detection probe-target complex becomes labeled with the first enzyme; contacting the biological sample with a first member of a first pair of click conjugates, the first member of the first pair of click conjugates comprising a tissue-reactive portion, wherein the first enzyme converts the first member of the first pair of click conjugates into a first reactive intermediate, the first member of the first pair of click conjugates comprising a tissue-reactive portion. covalently bonding a reactive intermediate to a biological sample proximate to or directly on a first target to form a first fixed tissue-click conjugate complex; contacting the biological sample with a second member of a first pair of click conjugates, the second member of the first pair of click conjugates comprising a second reactive moiety capable of reacting with the first reactive moiety of the first fixed tissue-click conjugate complex such that a covalent bond is formed between the first fixed tissue-click conjugate complex and the second member of the first pair of click conjugates to form a first tissue-click conjugate adduct; and detecting a signal from the first reporter moiety of the first tissue-click conjugate adduct.

[0122] In some embodiments, the second member of the first pair of click conjugates comprises at least one chromophore. In some embodiments, the first member of the first pair of click conjugates comprises a quinone methide precursor portion; and wherein the second member of the first pair of click conjugates comprises a chromophore. In some embodiments, the first member of the first pair of click conjugates comprises a tyramide portion; and wherein the second member of the first pair of click conjugates comprises a chromophore. In some embodiments, the first detection probe is a primary antibody, and wherein the first label conjugate comprises an anti-antibody antibody. In some embodiments, wherein the first enzyme is selected from the group consisting of a phosphatase, a phosphodiesterase, an esterase, a lipase, an amidase, a protease, a nitroreductase, a urease, a sulfatase, a cytochrome P450, an α-glucosidase, a β-glucosidase, a β-lactamase, an α-glucuronidase, a β-glucuronidase, an α-5-galactosidase, a neuraminidase, a β-galactosidase, an α-lactosidase, and a β-lactosidase.

[0123] In some embodiments, the method further comprises detecting a second target in the biological sample, wherein the second target is detected by: contacting the biological sample with a second detection probe specific for the second target to form a second detection probe-target complex; contacting the biological sample with a second labeled conjugate specific for the second detection probe, the second labeled conjugate comprising a second enzyme, such that the second detection probe-target complex becomes labeled with the second enzyme; contacting the biological sample with a first member of a second pair of click conjugates, the first member of the second pair of click conjugates comprising a tissue-reactive portion, wherein the second enzyme converts the first member of the second pair of click conjugates into a second reactive intermediate The invention also provides a method for preparing a biological sample comprising: contacting the biological sample with a second member of a second pair of click conjugates, wherein the second reactive intermediate is covalently bonded to a second target in proximity to or directly on the second target to form a second fixed tissue-click conjugate complex; contacting the biological sample with a second member of a second pair of click conjugates, the second member of the second pair of click conjugates comprising a second reactive moiety that is capable of reacting with the first reactive moiety of the second fixed tissue-click conjugate complex such that a covalent bond is formed between the second fixed tissue-click conjugate complex and the second member of the second pair of click conjugates; and detecting a signal from a second reporter moiety of the second tissue-click conjugate adduct, wherein the second reporter moiety is different from the first reporter moiety.

[0124] In some embodiments, the second member of the second pair of click conjugates comprises at least one chromophore. In some embodiments, the first member of the second pair of click conjugates comprises a quinone methide precursor portion; and wherein the second member of the second pair of click conjugates comprises a chromophore. In some embodiments, the first member of the second pair of click conjugates comprises a tyramide portion; and wherein the second member of the second pair of click conjugates comprises a chromophore. In some embodiments, the second detection probe is a primary antibody, and wherein the second first label conjugate comprises an anti-antibody antibody. In some embodiments, the second enzyme is selected from the group consisting of a phosphatase, a phosphodiesterase, an esterase, a lipase, an amidase, a protease, a nitroreductase, a urease, a sulfatase, a cytochrome P450, an α-glucosidase, a β-glucosidase, a β-lactamase, an α-glucuronidase, a β-glucuronidase, an α-5-galactosidase, a β-galactosidase, an α-lactase, and a β-lactase.

[0125] Another aspect of the present disclosure is an immobilized click conjugate covalently bonded to a tissue sample, the immobilized click-conjugate comprising a first reactive functional group selected from the group consisting of: dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine. In some embodiments, the click-conjugate is bonded to the tissue via a tyrosine residue or a nucleophile within or on the surface of the tissue sample.

[0126] In another aspect of the disclosure are detectable tissue-click adduct complexes formed by reacting an immobilized click-conjugate (such as shown above) with a conjugate of formula (IV):

[0127]

[0128] in

[0129] A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine and hydroxylamine;

[0130] A "linker" is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and

[0131] Z is selected from the group consisting of a chromophore, a fluorophore, an enzyme, a hapten, and a chelator; and

[0132] Wherein the conjugate of formula (IV) comprises an A group capable of reacting with the first reactive functional group of the immobilized Click-conjugate.

[0133] In some embodiments, Z is at least one chromophore. In some embodiments, the first reactive functional group is dibenzocyclooctyne, and wherein A of formula (IV) is selected from azide or 1,3-nitrone. In some embodiments, the first reactive functional group is trans-cyclooctene, and wherein A of formula (IV) is tetrazine. In some embodiments, the first reactive functional group is azide, and wherein A of formula (IV) is dibenzocyclooctyne. In some embodiments, Z is a chelator, and wherein a lanthanide is introduced into the detectable tissue-click adduct complex formed.

[0134] The applicants have found that the click conjugates disclosed herein are suitable for bioassays and that their use improves upon the limitations of TSA and QMSA. For example, and without wishing to be bound by any particular theory, the applicants have found that the quality of staining produced by QMSA is highly dependent on the solubility of the quinone methide-reporter conjugate. For example, very hydrophobic QMSA conjugates tend to stain discretely but with low signal intensity, while very hydrophilic QMSA conjugates tend to stain with high signal intensity but also with undesirable levels of diffusion. To address this issue, each quinone methide-reporter conjugate must be synthesized differently to optimize diffusion and signal intensity. For example, a very hydrophobic reporter may require an amphiphilic PEG linker, while a hydrophilic reporter may require a hydrophobic aliphatic linker. Therefore, QMSA can be tedious, and in some cases, the conjugate cannot be optimized with respect to limiting diffusion to a desired level. The applicants have found that amplification methods based on QMSA but utilizing the click conjugates herein improve upon QMSA.

[0135] Similarly, about TSA and again do not wish to be bound by any particular theory, the applicant believes that many fluorophores and chromophores are sensitive to oxidation, resulting in irreversible decomposition and loss of color and fluorescence. The applicant also believes that the oxidation conditions required for TSA determination can accelerate the oxidation of some dyes (i.e., cyanine dyes), making them poor reporters for TSA (although the substrate concentration is high, the signal intensity is low). The applicant has also shown that the hydrophobicity of tyrosine amide coupled with the hydrophobicity of many fluorophores and chromophores can cause tyrosine amide conjugates to be insoluble in the required IHC and ISH aqueous reaction medium. For almost all conjugates, amphiphilic PEG linkers are needed to dissolve the conjugate. However, in some cases, PEG linkers are not enough to overcome hydrophobicity, making some desired reporters (i.e., dabsyl) unavailable. Due to these two limitations, two desired color spaces, i.e., blue (cy5) and yellow (dabsyl) are currently unavailable using TSA. The applicant has found that the amplification method based on TSA but utilizing the click conjugates herein has improved TSA.

[0136] A further limitation common to both QMSA and TSA is the inability to amplify signals above a certain saturation point. There are a finite number of reactive sites on tissue to which these QMSA and TSA reactive intermediates bind. Once those reactive sites are depleted, the signal intensity saturates. Applicants have discovered that amplification methods utilizing the click conjugates and methods disclosed herein can increase the overall signal, thereby allowing visualization of low-abundance markers.

[0137] The applicants have surprisingly discovered that amplification using click conjugate pairs as described herein: (i) "shields" the reporter molecule (here, the component part of one member of a pair of click conjugates) from potentially unfavorable conditions (i.e., in the case of TSA, oxidatively unstable chromophores), allowing the use of a wider range of reporters; (ii) solves the water solubility issues associated with some tyramide conjugates of TSA (i.e., dabsyl); and (iii) for QMSA, only the solubility of a few QM-"click" conjugates needs to be "tuned," rather than an entire library of fluorophores, chromophores, and haptens. The applicants believe that these properties together provide simplified amplification methods for both QMSA and TSA, the ability to add additional desired color spaces to the TSA palette, and an overall enhancement of staining intensity. These and other aspects are further described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided to the Office upon request and payment of the necessary fee.

[0140] Figure 1A and 1B Reaction schemes illustrating the reaction between certain click conjugates comprising a quinone methide precursor moiety and a tissue-bound enzyme; followed by a reaction between the resulting tissue-click conjugate complex and a second click conjugate to form a tissue-click conjugate adduct are depicted.

[0141] Figure 2A and 2B Reaction schemes illustrating the reaction between certain click conjugates containing a tyramide moiety and a tissue-bound enzyme; followed by a reaction between the resulting tissue-click conjugate complex and a second click conjugate to form a tissue-click conjugate adduct are depicted.

[0142] Figure 3A Examples of first and second members of a pair of click conjugates are described, wherein the first member of each pair of click conjugates comprises a compound of formula (II) as described herein.

[0143] Figure 3B Examples of first and second members of a pair of click conjugates are described, wherein the first member of each pair of click conjugates comprises a compound of formula (III) as described herein.

[0144] Figure 4 Examples of click conjugates comprising at least one chromophore and a reactive functional group are described.

[0145] Figure 5A reaction between a tissue-bound quinone methide-containing click conjugate (tissue-click conjugate complex) and a click conjugate comprising at least one reporter moiety is described.

[0146] Figure 6 A reaction between a tyramide-containing click conjugate bound to tissue (tissue-click conjugate complex) and a click conjugate comprising at least one reporter moiety is described.

[0147] Figure 7A 、 7B and 7C illustrate staining of tonsil samples using certain QMSA conjugates.

[0148] Figure 8A 、 8B and 8C illustrate staining of tonsil samples with certain TSA conjugates.

[0149] Figure 9 A, 9B, 9C and 9D comparatively illustrate staining with different click conjugates with different reporter moieties.

[0150] Figure 10 The results of IHC staining using (i) a DAB control, (ii) using a TSA protocol, and (iii) using an amplification protocol utilizing click-conjugates of the present disclosure are comparatively illustrated.

[0151] Figure 11 The difference in staining intensity in ISH assays using (i) a TSA protocol and (ii) an amplification protocol utilizing the click conjugates of the present disclosure is comparatively illustrated.

[0152] Figure 12 The difference in staining intensity when using a click conjugate comprising a single chromophore and a click conjugate comprising multiple chromophores is comparatively illustrated.

[0153] Figure 13 Illustrated are the staining intensity using click conjugates containing alkaline phosphatase as the reporter moiety.

[0154] Figure 14 A reaction scheme is depicted illustrating the reaction between a tyramide-containing click conjugate and a tissue-bound enzyme; followed by a reaction between the resulting tissue-click conjugate complex and a second click conjugate comprising an alkaline phosphatase reporter moiety.

[0155] Figure 15 A flow chart depicting the steps for detecting a target within a biological sample using an amplification protocol utilizing the click conjugates of the present disclosure is set forth.

[0156] Details

[0157] In general, the present disclosure relates to click conjugates and methods for using click conjugates to detect one or more targets present in a biological sample. In some embodiments, the click conjugates (or kits comprising one or more click conjugates) are used in multiplex assays to simultaneously or sequentially detect multiple targets within a tissue sample. These and other aspects of the present disclosure are described in more detail herein.

[0158] definition

[0159] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" is defined in an inclusive manner such that "comprising A or B" means including A, B, or A and B.

[0160] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Specifically, each term is defined consistently with the customary U.S. patent law definition of "comprising," and is therefore to be interpreted as an open term meaning "at least the following," and is also to be interpreted as not excluding additional features, limitations, aspects, and the like. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Furthermore, while the steps and methods may be outlined herein in a particular order, a skilled artisan will recognize that the ordering of the steps and methods may vary.

[0161] As used herein, alkaline phosphatase (AP) is an enzyme that removes and transfers phosphate groups from organic esters (via hydrolysis) by breaking the phosphate-oxygen bond and temporarily forming an intermediate enzyme-substrate bond. For example, AP hydrolyzes naphthol phosphate (substrate) into phenolic compounds and phosphoric acid. Phenolic compounds are coupled with colorless diazonium salts (chromogens) to produce insoluble colored azo dyes.

[0162] As used herein, the term "antibody", occasionally abbreviated as "Ab", refers to immunoglobulins or immunoglobulin-like molecules, including, for example, but not limited to, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules and antibody fragments produced during an immune response in any vertebrate (e.g., in mammals such as humans, goats, rabbits and mice), which specifically bind to a target molecule (or a group of highly similar target molecules) to the substantial exclusion of binding to other molecules. Antibodies further refer to polypeptide ligands comprising at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. Antibodies can be composed of heavy and light chains, each having a variable region, referred to as a variable heavy (VH) region and a variable light (VL) region. The VH and VL regions are jointly responsible for binding to the antigen recognized by the antibody. The term antibody also includes complete immunoglobulins and variants and portions thereof well known in the art.

[0163] As used herein, the phrase "antibody conjugate" refers to those antibodies that are conjugated (directly or indirectly) to one or more markers, wherein the antibody conjugate is specific for a particular target and wherein the marker can be detected (directly or indirectly), such as with a secondary antibody (anti-marker antibody). For example, the antibody conjugate can be coupled to a hapten, such as through a polymer linker and / or a spacer, and the antibody conjugate can be detected indirectly with the aid of the hapten. As an alternative example, the antibody conjugate can be coupled to a fluorophore, such as through a polymer linker and / or a spacer, and the antibody conjugate can be detected directly. Antibody conjugates are further described in U.S. Publication No. 2014 / 0147906 and U.S. Patent 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 those antibodies that are conjugated to an enzyme, such as HRP or AP.

[0164] As used herein, the term "antigen" refers to a compound, composition or substance that can be specifically bound by a product of specific humoral or cellular immunity, such as an antibody molecule or a T cell receptor. 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.

[0165] As used herein, the term "biological sample" can be any solid or fluid sample obtained from, discharged or secreted by any living organism, including but not limited to unicellular organisms, especially such as bacteria, yeast, protozoa and amoeba, and multicellular organisms (such as plants or animals, including samples from healthy or apparently healthy human subjects or human patients affected by conditions to be diagnosed or investigated or diseases such as cancer). For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous humor or vitreous humor or any body secretions, exudates, effluents (for example, fluid obtained from an abscess or any other infection or inflammation site), or fluid obtained from a joint (for example, a normal joint or a joint affected by a disease). A biological sample can also be a sample obtained from any organ or tissue (including biopsy or autopsy specimens, such as tumor biopsy), or can include cells (whether primary cells or cultured cells) or a culture medium regulated by any cell, tissue or organ. In some instances, a biological sample is a nuclear extract. In some instances, the sample is a quality control sample, such as one of the disclosed cell mass slice samples. In other instances, the sample is a test sample. Those of ordinary skill in the art can use any method known in the art to prepare the sample. The sample can be obtained from a subject for routine screening or from a subject suspected of having a disease, such as a genetic abnormality, infection or neoplasia. The described embodiments of the disclosed methods can also be applied to samples that do not have a genetic abnormality, disease, disease, etc., which are referred to as "normal" samples. The sample can include multiple targets that can be specifically bound by one or more detection probes.

[0166] As used herein, the term "chromophore" refers to a molecule or portion of a molecule that is responsible for its color. Color is produced when a molecule absorbs certain wavelengths of visible light and transmits or reflects other wavelengths. A molecule having an energy difference between two different molecular orbitals that fall within the visible spectrum can absorb visible light and is therefore appropriately characterized as a chromophore. Visible light incident on a chromophore can be absorbed, thereby exciting electrons from a ground-state molecular orbital to an excited-state molecular orbital.

[0167] As used herein, the term "conjugate" refers to two or more molecules or moieties (including macromolecules or supramolecular molecules) covalently linked into a larger construct. In some embodiments, the conjugate includes one or more biomolecules (such as peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) covalently linked to one or more other molecular moieties.

[0168] As used herein, the term "couple" or "coupling" refers to the joining, bonding (eg, covalent bonding), or linking of one molecule or atom to another molecule or atom.

[0169] As used herein, a "hapten" is a small molecule that can be specifically combined with an antibody but is generally not substantially immunogenic unless combined with a carrier molecule. In some embodiments, haptens include, but are not limited to, pyrazoles (e.g., nitropyrazoles); nitrophenyl compounds; benzofurans; triterpenes; ureas (e.g., phenylureas); thioureas (e.g., phenylthioureas); rotenone and rotenone derivatives; oxazoles (e.g., oxazolesulfonamides); thiazoles (e.g., thiazolesulfonamides); coumarin and coumarin derivatives; and cyclolignans. Additional non-limiting examples of haptens include thiazoles; nitroaryls; benzofurans; triterpenes; and cyclolignans. Specific examples of haptens include dinitrophenyl, biotin, digoxigenin, and fluorescein, and any derivatives or analogs thereof. Other haptens are described in US Patent Nos. 8,846,320; 8,618,265; 7,695,929; 8,481,270; and 9,017,954; the disclosures of which are incorporated herein by reference in their entireties. Haptens themselves may be suitable for direct detection, ie, they may emit a suitable signal for detection.

[0170] As used herein, horseradish peroxidase (HRP) is an enzyme that can be conjugated to the molecule of the label. When hatched together with appropriate substrates, it produces colored, fluorescent or luminescent derivatives of the molecule of the label, allowing it to be detected and quantified. HRP works in the presence of an electron donor, to first form an enzyme-substrate complex, and then subsequently works to oxidize the electron donor. For example, HRP can act on 3,3'-diaminobenzidine tetrahydrochloride (DAB) to produce detectable color. HRP can also act on the tyramide conjugate of the label, or tyramide-like reactive conjugates (i.e., ferulic acid, coumaric acid, caffeic acid, cinnamic acid, dopamine, etc.), to deposit colored or fluorescent or colorless reporter moieties for tyramide signal amplification (TSA).

[0171] As used herein, the terms "multiplex," "multiplexed," or "multiplexing" refer to the simultaneous, substantially simultaneous, or sequential detection of multiple targets in a sample. Multiplexing can include identifying and / or quantifying multiple different nucleic acids (e.g., DNA, RNA, mRNA, miRNA) and polypeptides (e.g., proteins), both individually and in any and all combinations.

[0172] 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 an immunohistochemistry procedure.

[0173] As used herein, a "quinone methide" is a quinone analog in which one of the carbonyl oxygens on the corresponding quinone is replaced by a methylene group (CH2) to form an alkene.

[0174] As used herein, the term "secondary antibody" refers herein to an antibody that specifically binds to a primary antibody, thereby forming a bridge between the primary antibody and subsequent reagents (e.g., labels, enzymes, etc., if any). The secondary antibody is typically the second antibody used in immunohistochemistry procedures.

[0175] As used herein, the term "specific binding moiety" refers to a member of a specific binding pair. A specific binding pair is a pair of molecules characterized in that they bind to each other to the substantial exclusion of binding to other molecules (e.g., a specific binding pair may have an association constant that is at least 10 greater than that of either member of the binding pair to other molecules in a biological sample). -3 M, Big 10 -4 M or Big 10 -5 Specific binding moieties include molecules (or portions thereof) that are specifically bound by such specific binding proteins.

[0176] As used herein, the term "target" refers to any molecule whose presence, location, and / or concentration is determined or can be determined. Examples of target molecules include proteins, nucleic acid sequences, and haptens, such as haptens covalently bonded to proteins. Target molecules are typically detected using one or more conjugates of a specific binding molecule and a detectable label.

[0177] "Click" conjugates

[0178] The present disclosure provides two general subsets of click conjugates. The first subset of click conjugates comprises a tissue-reactive portion coupled to a reactive functional group via an optional linker. In some embodiments, the first subset of click conjugates serves as the first member of a click conjugate pair. The second subset of click conjugates comprises one or more reporter portions coupled to a reactive functional group via an optional linker. In some embodiments, the second subset of click conjugates serves as the second member of a click conjugate pair. It should be understood that the different subsets of click conjugates disclosed herein can act as modular "building blocks" such that when any two conjugates having appropriate reactive functional groups are combined (a "pair of click conjugates"), they can undergo a reaction and form a covalent bond, thereby coupling the two conjugates to form a "click adduct" having a desired structure or component portion.

[0179] As will be further described herein, the click adducts formed can serve as substances suitable for detecting targets in biological assays. Without wishing to be bound by any particular theory, it is believed that the click conjugates disclosed herein are stable in aqueous media and are therefore suitable for use in certain biological assays, including IHC and ISH. In addition, it is believed that click conjugates have a large thermodynamic driving force, which is conducive to rapid reactions that provide a single product. In addition, the solubility of any click conjugate described herein can be "tuned" to meet the requirements of any particular assay, and such "tuning" can be achieved by, for example, introducing a water-soluble linker or a water-soluble linker component into the conjugate. In addition, reactions comprising the click conjugates described herein can be carried out in a wide variety of buffers and, therefore, can be performed at a wide variety of pHs, allowing the skilled person to select ideal conditions for reporter stability.

[0180] In one aspect of the disclosure are click conjugates of formula (I):

[0181]

[0182] wherein A is a reactive functional group, "linker" is an optional connecting group, and B is selected from a "tissue reactive moiety" or a reporter moiety.

[0183] As used herein, the term "tissue reactivity" refers to a part that can react with an enzyme. Therefore, when the click conjugate comprising a tissue reactivity portion reacts with an appropriate enzyme, the part of the tissue reactivity portion of the click conjugate undergoes structure, conformation and / or electronic changes, thereby providing a tissue reactivity substance (intermediate, including free radical intermediates) suitable for being directly or indirectly bonded to a biological sample (or to the extent possible, in a biological sample). For example, when the tissue reactivity portion is junket amide or a derivative thereof, when junket amide reacts with an appropriate enzyme (such as HRP), a junket amide free radical substance (intermediate) is formed. This highly reactive junket amide free radical substance can be bonded to the tyrosine residue in the biological sample. In a similar manner, the quinone methide precursor moiety, after reacting with an appropriate enzyme (such as AP), is converted into quinone methide, which is believed to react highly with the nucleophile in the biological sample. The effect of the part of the tissue reactivity portion of any click conjugate, its interaction with an appropriate enzyme, and the formation of immobilized tissue-click conjugate complex are further described herein.

[0184] In some embodiments, A is selected from dibenzocyclooctyne ("DBCO"), trans-cyclooctene ("TCO"), azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine. In some embodiments, A is selected from a group capable of undergoing a photo-initiated reaction.

[0185] The click conjugate optionally comprises a "linker". In some embodiments, a "linker" is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, a "linker" comprises one or more groups selected from amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl and imine groups. The linker chain may also comprise an aromatic group, including a heteroaromatic group, wherein the heteroaromatic group comprises 1 to 4 heteroatoms selected from O, N or S.

[0186] In some embodiments, the "linker" has the structure depicted in Formula (Ia):

[0187]

[0188] wherein d and e are each independently an integer ranging from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S or N (R c )(R d );R a and R b are independently H, C1-C4 alkyl, F, Cl or N(R c )(R d );R c and R d is independently CH3 or H; and X and Y are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally having one or more O, N or S heteroatoms. In some embodiments, X and Y include carbonyl groups, amide groups, ester groups, ester groups, substituted or unsubstituted aryl groups or any combination thereof. In other embodiments, d and e are integers ranging from 2 to 10. In yet other embodiments, d and e are integers ranging from 2 to 6.

[0189] In some embodiments, the "linker" has the structure depicted in Formula (Ib):

[0190]

[0191] in

[0192] d and e are each independently an integer ranging from 2 to 20;

[0193] t and u are independently 0 or 1;

[0194] Q is a bond, O, S or N (R c )(R d );

[0195] R c and R dare independently CH3 or H; and

[0196] X and Y are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally having one or more O, N or S heteroatoms.

[0197] In some embodiments, X and Y are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 2 to 8 carbon atoms and optionally having one or more O, N, or S heteroatoms.

[0198] In some embodiments, the "linker" has the structure depicted in Formula (Ic):

[0199]

[0200] in

[0201] d and e are each independently an integer ranging from 2 to 20;

[0202] t and u are independently 0 or 1; and

[0203] X and Y are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally having one or more O, N or S heteroatoms.

[0204] In other embodiments, d and e are integers ranging from 2 to 10. In still other embodiments, d and e are integers ranging from 2 to 6.

[0205] The alkylene oxide-based "linkers" of formula (Ia), (Ib) and (Ic) are referred to herein by reference to glycols, such as ethylene glycols. In some embodiments, the incorporation of such alkylene oxide linkers is believed to increase the hydrophilicity of the click conjugates. One of ordinary skill in the art will understand that as the number of alkylene oxide repeating units in the linker increases, the hydrophilicity of the conjugate may also increase. Additional heterobifunctional polyalkylene glycol spacers that can be used to implement certain disclosed embodiments of the present disclosure are described in the assignee's co-pending applications, including U.S. Patent Application No. 11 / 413,778, filed April 28, 2006, for "Nanoparticle Conjugates"; U.S. Patent Application No. 11 / 413,415, filed April 27, 2006, for "Antibody Conjugates"; and U.S. Provisional Patent Application No. 60 / 739,794, filed November 23, 2005, for "Molecular Conjugates"; all of which are incorporated herein by reference.

[0206] Tissue-Reactive Promoiety "Click" Conjugates

[0207] In some embodiments, the click conjugates of the present disclosure have the structure of Formula (Id):

[0208]

[0209] wherein the "tissue-reactive precursor moiety" is (i) a tyramide or a derivative or analog thereof, or (ii) a quinone methide precursor; and wherein A and the linker are as defined above. Exemplary quinone methide precursor derivatives suitable for incorporation into the disclosed click conjugates of formula (I) include those listed in PCT / EP2015 / 053556, entitled "Quinone Methide Analog Signal Amplification," having an international filing date of February 20, 2015, the disclosure of which is hereby incorporated by reference in its entirety.

[0210] Quinone methide "click" conjugates

[0211] In some embodiments, the compound has formula (II):

[0212]

[0213] in

[0214] A is as defined above;

[0215] A "linker" is an optional linking group as defined above; and

[0216] U is a quinone methide precursor, a derivative or an analog thereof.

[0217] In some embodiments, the compound of Formula (II) is the first member of a pair of click conjugates.

[0218] As used herein, "quinone methide precursors" are a class of conjugated compounds that are converted to highly reactive quinone methides when reacted with an appropriate enzyme (e.g., AP). As indicated above, quinone methide precursors and their conversion to quinone methides are described in PCT / EP2015 / 053556, the disclosure of which is hereby incorporated by reference in its entirety.

[0219] In some embodiments, the quinone methide precursor portion of the conjugate of formula (II) is derived from one of the following quinone methide precursor derivatives:

[0220]

[0221]

[0222] In some embodiments, the conjugate of formula (II) has the structure of formula (IIa):

[0223]

[0224] wherein "Linker" and A are as defined herein,

[0225] R 1 is a group selected from a phosphate group, an amide group, a nitro group, a urea group, a sulfate group, a methyl group, an ester group, a β-lactam group or a sugar group;

[0226] R 2 It is a halogen;

[0227] R 3 、R 5 and R 6 independently selected from hydrogen or an aliphatic group having 1 to 4 carbon atoms;

[0228] R 4 is hydrogen, an aliphatic group having 1 to 4 carbon atoms or a group -CH(R 2 )-R 7 -[Connector]-A;

[0229] R 7 Yes - (CH2) w NH-, -O(CH2) w NH-, -N(H)C(O)(CH2) w NH-、-C(O)N(H)(CH2) w NH-, -(CH2) w O-, -O(CH2) w O-, -O(CH2CH2O) w -、-N(H)C(O)(CH2) w O-, -C(O)N(H)(CH2) w O-, -C(O)N(H)(CH2CH2O) w -、-(CH2) w S-, -O(CH2) w S-, -N(H)C(O)(CH2) w S-、-C(O)N(H)(CH2) w S-, -(CH2) w NH-, -C(O)N(H)(CH2CH2O) w CH2CH2NH, -C(O)(CH2CH2O) w CH2CH2NH-, -C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2) wNH- or -N(H)(CH2) w NH-, wherein w is an integer ranging from 1 to 12. When R1 is a sugar, the sugar may be selected from glucose, β-glucose, α-galactoside, β-galactoside, α-glucuronose, neuraminic acid or β-glucuronose.

[0230] In other embodiments, the conjugate of formula (II) has the structure of formula (lib):

[0231]

[0232] where R 1 is selected from phosphoric acid, amide, nitro, urea, sulfuric acid, methyl, ester, β-lactam or sugar; and

[0233] where R 7 Yes - (CH2) w NH-, -O(CH2) w NH-, -N(H)C(O)(CH2) w NH-、-C(O)N(H)(CH2) w NH-, -(CH2) w O-, -O(CH2) w O-, -O(CH2CH2O) w -、-N(H)C(O)(CH2) w O-, -C(O)N(H)(CH2) w O-, -C(O)N(H)(CH2CH2O) w -、-(CH2) w S-, -O(CH2) w S-, -N(H)C(O)(CH2) w S-、-C(O)N(H)(CH2) w S-, -(CH2) w NH-, -C(O)N(H)(CH2CH2O) w CH2CH2NH, -C(O)(CH2CH2O) w CH2CH2NH-, -C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2) w NH- or -N(H)(CH2) w NH-, wherein w is an integer ranging from 1 to 12.

[0234] In some embodiments of the conjugate of Formula (IIb), R 1 is phosphoric acid and R 7 is -C(O)N(H)(CH2) wNH-, and w ranges from 2 to 10.

[0235] In yet other embodiments, the conjugate of formula (II) has the structure of formula (IIc):

[0236]

[0237] where R 7 Yes - (CH2) w NH-, -O(CH2) w NH-, -N(H)C(O)(CH2) w NH-、-C(O)N(H)(CH2) w NH-, -(CH2) w O-, -O(CH2) w O-, -O(CH2CH2O) w -、-N(H)C(O)(CH2) w O-, -C(O)N(H)(CH2) w O-, -C(O)N(H)(CH2CH2O) w -、-(CH2) w S-, -O(CH2) w S-, -N(H)C(O)(CH2) w S-、-C(O)N(H)(CH2) w S-, -(CH2) w NH-, -C(O)N(H)(CH2CH2O) w CH2CH2NH, -C(O)(CH2CH2O) w CH2CH2NH-, -C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2) w NH- or -N(H)(CH2) w NH-, wherein w is an integer ranging from 1 to 12.

[0238] In some embodiments, R 7 is C(O)N(H)(CH2) w NH and w are as defined above. In other embodiments, R 7 is C(O)N(H)(CH2) w NH and w ranges from 2 to 6.

[0239] In yet a further embodiment, the conjugate of formula (II) has the structure of formula (IId):

[0240]

[0241] in

[0242] w ranges from 1 to 12, and

[0243] "Linker" and A are as defined above.

[0244] In some embodiments, w ranges from 1 to 8. In other embodiments, w ranges from 2 to 8. In still other embodiments, w ranges from 2 to 6. In further embodiments, w is 6.

[0245] Specific examples of the compound of formula (II) include the following:

[0246]

[0247] The quinone methide precursor click conjugates of formula (II) can be synthesized according to any method known to those of ordinary skill in the art. In some embodiments, a reagent comprising a desired reactive functional group and a linker is coupled only to the quinone methide precursor or a derivative or analog thereof, as illustrated in the following reaction scheme. For example, a quinone methide precursor having a terminal amine group can be coupled to a compound comprising an amine reactive group (e.g., an active ester such as N-hydroxysuccinimide (NHS) or sulfo-NHS, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, ethylene oxide, carbonate, aryl halide, imidate, anhydride, etc.).

[0248] In some of the specific examples below, a click partner with an NHS-ester group is coupled to a quinone methide precursor with a terminal amine. In some embodiments, the reaction occurs in DMSO and is allowed to react for 60 minutes. The reactants are then diluted with methanol and directly purified by preparative HPLC.

[0249]

[0250] Scheme 1A: Example of the synthesis of a compound of formula (II).

[0251] Tyramide "click" conjugates

[0252] In other embodiments, the compound has formula (III):

[0253]

[0254] wherein A is as defined above; "Linker" is an optional linking group as defined above; and M is tyramide or a derivative or analog thereof. In some embodiments, the compound of formula (III) is the first member of a pair of click partners.

[0255] In some embodiments, the conjugate of Formula (III) comprises a tyramide derived from a compound having the structure of Formula (Ilia):

[0256]

[0257] wherein each R group is independently selected from hydrogen or a lower alkyl group having 1 to 4 carbon atoms (which may be linear or branched), and wherein the linker and A are as defined herein.

[0258] In some embodiments, the compound of Formula (III) comprises a tyramide derived from a compound having the structure of Formula (IIIb):

[0259]

[0260] wherein A is selected from azide, thiol, 1,3-nitrone, hydrazine or hydroxylamine, and wherein the linker is as defined herein.

[0261] In some embodiments, the compound of Formula (III) comprises a moiety from a compound having the structure of Formula (IIIc) or (IIId):

[0262] .

[0263] Non-limiting examples of specific tyramide click conjugates include the following:

[0264]

[0265]

[0266] The junket amide click conjugates of formula (III) can be synthesized according to any method known to those of ordinary skill in the art. In some embodiments, the reagent comprising the desired reactive functional group and the joint is coupled only to junket amide or its derivatives or analogs, as illustrated in the following reaction scheme. For example, junket amide (having a terminal amine group) can be coupled to a compound comprising an amine-reactive group (e.g., an active ester such as N-hydroxysuccinimide (NHS) or sulfo-NHS, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, ethylene oxide, carbonate, aryl halide, imidate, anhydride, etc.).

[0267] In some of the specific examples below, a click partner with an NHS-ester group was coupled to a tyramide. In some embodiments, the reaction was carried out in DMSO and allowed to react for 60 minutes. The reactants were then diluted with methanol and directly purified by preparative HPLC.

[0268]

[0269] Scheme 1B: Example of the synthesis of a click conjugate of formula (III).

[0270] Reporter moiety "click" conjugate

[0271] In other embodiments, the click conjugates of the present disclosure have the structure of Formula (IV):

[0272]

[0273] wherein A is as defined above; "Linker" is an optional linking group as defined above; and Z comprises at least one reporter moiety (the terms "reporter moiety" and "reporter" are used interchangeably herein). In some embodiments, the compound of formula (IV) is the second member of a pair of click partners.

[0274] In some embodiments, the conjugate of formula (IV) comprises one reporter moiety, and thus group Z is a reporter moiety that is coupled directly or indirectly through a linker to the reactive functional group A. In other embodiments, the conjugate of formula (IV) comprises multiple reporter moieties, and thus Z represents a group having two or more reporter moieties. In embodiments where Z represents a group having two or more reporter moieties, group Z is coupled directly or indirectly through a linker to the reactive functional group A.

[0275] In some embodiments, Z comprises two reporters. In other embodiments, Z comprises four reporters. In other embodiments, Z comprises six reporters. In yet other embodiments, Z comprises more than six reporters. In embodiments in which Z comprises more than one reporter, the reporters can be the same or different. For example, Z can comprise two identical chromogens (e.g., two TAMRA chromogens). Alternatively, Z can comprise two different chromogens (e.g., TAMRA and cy5).

[0276] In some embodiments, Z comprises at least two reporter moieties, and the at least two reporter moieties are linked to each other via a linear or branched aliphatic group (optionally comprising one or more heteroatoms). In other embodiments, Z comprises at least two reporter moieties, and the at least two reporter moieties are linked to each other via a dendrimer or branched polymer.

[0277] In some embodiments, the compound of Formula (IV) has the structure of Formula (V):

[0278]

[0279] in

[0280] A "scaffold" is a group capable of coupling multiple reporter moieties, and

[0281] v is an integer ranging from 1 to 20.

[0282] In some embodiments, the "scaffold" is a polyamine (e.g., norispermidine, spermine, and derivatives or analogs thereof; or a polyamine containing 2 to 10 amine groups); a heterobifunctional linker (e.g., lysine or a lysine derivative); a dendrimer (e.g., polyamidoamine (PAMAM) dendrimers, Janus dendrimers (i.e., dendrimers composed of two dendritic wedges and terminated by two different functional groups), and bis-MPA dendrimers and derivatives thereof); or a polymer. In some embodiments, the "scaffold" is a bond.

[0283] In some embodiments, the click conjugate has the formula:

[0284]

[0285]

[0286] wherein Linker and Z are as defined herein.

[0287] In some embodiments, the reporter moiety is selected from a chromophore, a fluorophore, an enzyme, a hapten, or a chelator.

[0288] Non-limiting examples of suitable haptens include pyrazoles, particularly nitropyrazoles; nitrophenyl compounds; benzofurazans; triterpenes; ureas and thioureas, particularly phenylureas, and even more particularly phenylthioureas; rotenone and rotenone derivatives, also referred to herein as rotenones; oxazoles and thiazoles, particularly oxazole and thiazole sulfonamides; coumarins and coumarin derivatives; cyclolignans, such as podophyllotoxin and podophyllotoxin derivatives; and combinations thereof. Further examples of haptens and methods of synthesizing and using them are disclosed in U.S. Pat. No. 7,695,929, the disclosure of which is hereby incorporated by reference in its entirety.

[0289] In some embodiments, suitable haptens include BD (benzodiazepine), BF (benzofurazan), DABSYL (4-(dimethylamino)azobenzene-4'-sulfonamide, which has a maximum of about 436 nm), DCC (7-(diethylamino)coumarin-3-carboxylic acid), DIG (digoxigenin), DNP (dinitrophenyl), HQ (3-hydroxy-2-quinoxaline urea), NCA (nitrocinnamic acid), NP (nitropyrazole), PPT (podophyllotoxin), Rhod (rhodamine), ROT (rotenone), and TS (thiazolesulfonamide). Other suitable haptens include biotin and fluorescein derivatives (FITC (fluorescein isothiocyanate)), TAMRA (tetramethylrhodamine), Texas Red), and Rhodamine 110 (rhodamine).

[0290] Non-limiting examples of suitable chromophores include coumarin and coumarin derivatives.

[0291] Examples of coumarin-based chromophores include DCC and 2,3,6,7-tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizine-10-carboxylic acid. Other suitable chromophores include diazo-containing chromogens such as tartrazine. Still other suitable chromophores include triarylmethanes, including those provided below:

[0292] .

[0293] Additional non-limiting examples of suitable chromophores include those provided below:

[0294]

[0295] Other suitable chromophores include cyclic chromophores, such as those provided below:

[0296]

[0297]

[0298] Fluorophores belong to several common chemical classes, including coumarins, fluoresceins (or fluorescein derivatives and analogs), rhodamines, resorufins, luminophores, and cyanines. Additional examples of fluorescent molecules can be found in Molecular Probes Handbook — A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, OR, TheroFisher Scientific, 11th edition. In other embodiments, the fluorophore is selected from xanthene derivatives, cyanine derivatives, squaric acid derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, and tetrapyrrole derivatives. In other embodiments, the fluorescent moiety is selected from the group consisting of 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), SureLight dyes (available from APC, RPEPerCP, Phycobilisomes) (Columbia Biosciences), and APC, APCXL, RPE, BPE (available from Phyco-Biotech, Greensea, Prozyme, Flogen).

[0299] 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 comprises an oxidoreductase or a peroxidase (e.g., HRP, AP). Reference is made to Figure 14The use of an enzyme as a reporter moiety is further illustrated. The second member of a pair of click conjugates comprises a compound of formula (IV), wherein Z is an enzyme, particularly alkaline phosphatase. As will be further understood herein, the resulting click adduct can be detected by introducing an additional alkaline phosphatase reporter (chromogen, fluorophore).

[0300] In some embodiments, the reporter moiety is a chelating agent or chelating agent that can chelate in the presence of a lanthanide (e.g., europium). Without wishing to be bound by any particular theory, it is believed that lanthanide atoms can be detected using inductively coupled plasma mass spectrometry imaging (ICP-MSI). In addition, lanthanides can be detected using time-resolved fluorescence microscopy, which takes advantage of the relatively long lifetime of lanthanide luminescence compared to conventional fluorophores. In order to visualize lanthanides, an antenna ligand must be present to absorb and transfer energy to the lanthanide, which is typically poorly absorbed. The reaction product of the DBCO-azide click reaction can act as an antenna ligand, greatly simplifying the design of these systems.

[0301] An example of a compound of formula (IV) comprising an azide-reactive group conjugated to a chelator moiety is shown below:

[0302] .

[0303] In some embodiments, the reporter portion (Z) of the click conjugate of formula (IV) is selected from the group consisting of:

[0304]

[0305] In some embodiments, the compound of formula (IV) comprises the formula (IVa):

[0306]

[0307] wherein A is as described above. Although Formula (IVa) depicts the compound as comprising a PEG linker, other suitable linkers may be substituted.

[0308] In some embodiments, the compound of Formula (IV) comprises the formula (IVb):

[0309]

[0310] wherein A is as described above. Although Formula (IVb) depicts the compound as comprising a PEG linker, other suitable linkers may be substituted.

[0311] In some embodiments, the compound of formula (IV) comprises the formula (IVc):

[0312]

[0313] wherein A is as described above. Although Formula (IVc) depicts the compound as comprising a PEG linker, other suitable linkers may be substituted.

[0314] In some embodiments, the compound of formula (IV) comprises the formula (IVd):

[0315]

[0316] wherein A is as described above. Although Formula (IVd) depicts the compound as comprising a PEG linker, other suitable linkers may be substituted.

[0317] Specific non-limiting examples of conjugates of formula (IV) include the following:

[0318]

[0319]

[0320] Non-limiting examples of conjugates of formula (V) are illustrated below:

[0321] .

[0322] The reporter moiety compound of formula (IV) can be synthesized according to methods known to those of ordinary skill in the art. Examples of synthetic methods for coupling NHS esters to amines. This procedure can be applied to the reaction of any tyramide or quinone methide precursor containing an amine or NHS functional group with a click partner containing an amine or NHS ester functional group. It can also be applied to the reaction of a reporter group (chromogen, hapten, etc.) containing an amine or NHS functional group with a click partner containing an amine or NHS ester functional group.

[0323] Junk amide-peg5-DBCO. Junk amide (1.1 equivalents, 110 mg, 0.79 mmol) was dissolved in DMSO (3 mL), followed by the addition of triethylamine (5.0 equivalents, 360 mg, 3.6 mmol). DBCO-peg5-DBCO (1.0 equivalents, 500 mg, 0.72 mmol) was then added, and the resulting reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with MeOH (2 mL), and the resulting mixture was purified by preparative RP-HPLC (C18; 40 mL / min; 0.05% TFA in H2O:MeCN 95:5 to 5:95, over 40 minutes) to obtain Junk amide-peg5-DBCO (450 mg, 87% yield) as a colorless glass after removing the solvent under high vacuum. MS (ESI) m / z (M+H)+ calcd for C40H50N3O9+ 716.4, found 716.6.

[0324]

[0325] Coupling of click conjugate pairs

[0326] The skilled artisan will recognize that the click conjugates disclosed herein are suitable for coupling with each other to form "click adducts". The skilled artisan will also recognize that in order for one member of a pair of click conjugates to react with the other member of the pair of click conjugates and thereby form a covalent bond, both members of the pair of click conjugates must have reactive functional groups that are capable of reacting with each other. The table below illustrates different pairs of reactive functional groups that will react with each other to form a covalent bond.

[0327] Reactive functional groups on the first member of a pair of click conjugates Reactive functional groups on the second member of a pair of click conjugates DBCO Azide Olefins Tetrazine TCO Tetrazine Maleimide thiols DBCO 1,3-Nitrone Aldehyde or ketone Hydrazine Aldehyde or ketone Hydroxylamine Azide DBCO Tetrazine TCO thiols Maleimide 1,3-Nitrone DBCO Hydrazine Aldehyde or ketone Hydroxylamine Aldehyde or ketone Tetrazine Olefins

[0328] Specific non-limiting examples of click conjugate pairs having these reactive functional groups are illustrated in Figures 3 and 4. In particular, Figure 3 provides examples of click conjugate pairs, wherein one member of each pair comprises a compound of Formula (II). Figure 4 Also provided are examples of click conjugate pairs, wherein one member of each pair of click conjugates comprises a compound of formula (III).

[0329] In some embodiments, the click conjugates are coupled via a "strain-promoted azide-alkyne cycloaddition" (SPAAC) or a "TCO-tetrazine ligation" (TTL). SPAAC involves a reaction between an azide and a strained alkyne, whose high energy allows a 1,3-dipolar cycloaddition to occur in the absence of a Cu(I) catalyst (required for traditional azide-alkyne "click" chemistry). In some embodiments, dibenzocyclooctyne is used as the strained cyclooctyne due to its commercial availability and literature precedent. TTL utilizes the reaction between trans-cyclooctene and tetrazine to form a dihydropyridazine bond. These reagents are also commercially available and have been shown to react orthogonally with the SPAAC system.

[0330] The following schematics further illustrate the coupling of a pair of click conjugates containing different reactive functional groups. In the following scheme, one member of the pair of click conjugates is provided as an immobilized tissue-click conjugate complex. As will be further described herein, the immobilized tissue-click conjugate complex is formed by reacting a click conjugate of formula (II) or (III) with an appropriate enzyme and subsequently coupling the resulting reactive intermediate to the tissue.

[0331] For example, Scheme 2 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group and a second click conjugate of formula (IV) comprising a reactive azide group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined by a scaffold (e.g., a lysine linker or a dendrimer). In some embodiments, at least one reporter moiety Z is a chromophore. In some embodiments, at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.

[0332]

[0333] Similarly, Scheme 3 illustrates the reaction between an immobilized tissue-click conjugate complex having a TCO-reactive functional group and a second click conjugate of formula (IV) comprising a reactive tetrazine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined by a scaffold (e.g., a lysine linker or a dendrimer). In some embodiments, at least one reporter moiety Z is a chromophore. In some embodiments, at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.

[0334]

[0335] Scheme 4 again illustrates the reaction between an immobilized tissue-click conjugate complex having a maleimide reactive functional group and a second click conjugate of formula (IV) comprising a reactive thiol group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined by a scaffold (e.g., a lysine linker or a dendrimer). In some embodiments, at least one reporter moiety Z is a chromophore. In some embodiments, at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.

[0336]

[0337] Scheme 5 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group and a second click conjugate of formula (IV) comprising a reactive 1,3-nitrone group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined by a scaffold (e.g., a lysine linker or a dendrimer). In some embodiments, at least one reporter moiety Z is a chromophore. In some embodiments, at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.

[0338]

[0339] Scheme 6 illustrates the reaction between an immobilized tissue-click conjugate having an aldehyde-reactive functional group and a second click conjugate of formula (IV) comprising a reactive hydrazine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined by a scaffold (e.g., a lysine linker or a dendrimer). In some embodiments, at least one reporter moiety Z is a chromophore. In some embodiments, at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.

[0340]

[0341] Scheme 7 illustrates the reaction between an immobilized tissue-click conjugate having an aldehyde-reactive functional group and a second click conjugate of formula (IV) comprising a reactive hydroxylamine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined by a scaffold (e.g., a lysine linker or a dendrimer). In some embodiments, at least one reporter moiety Z is a chromophore. In some embodiments, at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.

[0342]

[0343] Scheme 8 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO-reactive functional group and a second click conjugate of formula (IV) comprising a reactive azide group and a chelator as reporter Z. In some embodiments, the resulting intermediate adduct comprises a chelator that, upon introduction of a lanthanide, forms a chelated adduct complex suitable for detection by MSI.

[0344]

[0345] Scheme 9A illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO-reactive functional group and a second click conjugate of Formula (IV) or Formula (V) comprising a reactive azide group coupled to a dendrimer, which is coupled to two, four, or eight reporter moieties, as shown. Without wishing to be bound by any particular theory, it is believed that the use of dendrimers allows the incorporation of multiple reporters (which can be the same or different), thereby providing significant signal amplification.

[0346]

[0347] Scheme 9B illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO-reactive functional group and a second click conjugate of formula (V) comprising a reactive azide group coupled to a dendrimer (PAMAM) to which are coupled four reporter moieties Z.

[0348]

[0349] Scheme 10 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO-reactive functional group and a second click conjugate of formula (V) comprising a reactive azide group coupled to a Z group comprising two chromophores, wherein the two chromophores are linked via a lysine group. Although the chromogens are described as being the same, the skilled artisan will recognize that the chromogens linked via the lysine groups can be different.

[0350]

[0351] Specific examples of fixed tissue-click conjugate complexes and their reactions with click conjugates containing specific reporter moieties are shown in Figure 5 and 6 middle.

[0352] Methods for detecting a target in a sample using click conjugates

[0353] The present disclosure also provides methods for detecting one or more targets within a tissue sample using a pair of any click conjugates. Although certain disclosed embodiments, examples, or figures herein may relate to the use of click conjugates in conjunction with IHC assays, the skilled artisan will appreciate that click conjugates can also be used in in situ hybridization (ISH) assays or any combination of IHC and ISH assays. The skilled artisan will also appreciate that click conjugates can be used in both simple and multiplex assays.

[0354] The methods described herein relate to click conjugate pairs suitable for use in biological assays. In those assays, one member (or "partner") of a particular pair of click conjugates comprises a conjugate of Formula (II) or (III), and the other member of the pair comprises a conjugate of Formula (IV) or (V). Typically, a first member of a pair of click conjugates is covalently deposited onto tissue using QMSA or TSA. Then, a second member of the pair of click conjugates, comprising a reporter molecule (i.e., a chromophore, fluorophore, enzyme, hapten), is applied to the tissue. The "click" reaction between the two "click" partners occurs rapidly, covalently binding the reporter molecule to the tissue at a location determined by the QMSA or TSA chemistry. In addition, and as shown herein, the amplification methods disclosed herein allow the reporter moiety to be separated from the QMSA or TSA assay conditions, which is believed to enhance signal intensity.

[0355] For example, Figure 1A 、 1B Figures 2A and 2B illustrate the reaction between the first member of a pair of click conjugates (10, 20) having a tissue-reactive moiety and a target-bound enzyme (11, 21) to form an immobilized tissue-click conjugate complex (13, 23). This first part of the amplification process is similar to that used in QMSA and TSA amplification processes. Figure 1A 、 1B , 2A and 2B also illustrate the subsequent reaction between the fixed tissue-click conjugate (13, 23) complex and the second member of the pair of click conjugates (14, 24) to provide an fixed tissue-click adduct complex (15, 25) comprising a detectable reporter moiety.

[0356] refer to Figure 1A , a compound (10) of formula (II) comprising a reactive functional group is contacted with a target-bound enzyme (11) to produce an active intermediate (12). In this example, the reactive intermediate (quinone methide) forms a covalent bond with a nucleophile on or within the tissue sample, thereby providing an immobilized tissue-click conjugate complex (13). The immobilized tissue-click conjugate complex can then be reacted with a compound (14) of formula (IV), provided that click conjugate 10 and click conjugate 14 have reactive functional groups that can react with each other to form a covalent bond. The reaction product of the immobilized tissue-click conjugate complex 13 and click conjugate 14 produces an immobilized tissue-click adduct complex 15. The tissue-click adduct complex 15 can be detected by means of a signal transmitted from a linked reporter moiety. In some embodiments, the reporter moiety is at least one chromophore.

[0357] Figure 1BThe reaction between a specific compound of formula (II) having a quinone methide precursor moiety attached to a DBCO reactive functional group and a target-bound enzyme is illustrated to produce a reactive quinone methide intermediate, which is then coupled to a nucleophile on or within a biological sample. More specifically, alkaline phosphatase recognizes and cleaves the phosphate group of the quinone methide precursor moiety shown from the click conjugate, resulting in the ejection of a leaving group and the formation of a corresponding quinone methide intermediate. The fixed tissue-click conjugate can then react with a compound of formula (IV), such as a compound comprising an azide group and a chromophore, as illustrated. The resulting product is a tissue-click adduct complex having a detectable chromophore as depicted.

[0358] Similarly, and with reference Figure 2A , a compound (20) of formula (III) containing a reactive functional group is contacted with a target-bound enzyme (21) to produce a reactive intermediate (22), i.e., a tyramide radical species (or its derivative). The tyramide radical intermediate can then form a covalent bond with the tissue sample, thereby providing a fixed tissue-click conjugate complex (23). The fixed tissue-click conjugate complex can then react with a compound (24) of formula (IV), provided that click conjugates 20 and 24 have reactive functional groups that can react with each other to form covalent bonds. The reaction product of the fixed tissue-click conjugate complex 23 and click conjugate 24 produces a tissue-click adduct complex 25.

[0359] Figure 2B Reactions between specific compounds of formula (III), ie, compounds having a tyramide moiety attached to a DBCO-reactive functional group, are illustrated. Figure 4 The target-bound enzyme is also described to produce a reactive tyramide radical intermediate, which is then coupled to a biological sample to form an immobilized tissue-click conjugate complex. The immobilized tissue-click conjugate complex can then be reacted with a compound of formula (IV), such as a compound comprising an azide group and a chromophore, as described. The resulting product is a tissue-click adduct complex (25) having a detectable chromophore.

[0360] In some embodiments, the method for detecting a target in a biological sample comprises the following steps: first, contacting the biological sample with a first detection probe specific for a first target. The first detection probe can be a primary antibody or a nucleic acid probe. Subsequently, contacting the sample with a first labeled conjugate, the first labeled conjugate comprising a first enzyme. In some embodiments, the first labeled conjugate is a secondary antibody specific for a primary antibody or a marker conjugated to a nucleic acid probe. Next, contacting the biological sample with a first member of a pair of click conjugates, the first member of the pair of click conjugates having a structure of any one of the compounds of formula (II) or (III). As described herein, the first enzyme cleaves the first member of the pair of click conjugates, thereby converting the first member into a reactive intermediate that covalently binds the biological sample close to the target or directly on the target. Next, introducing the second member of the pair of click conjugates, the second member of the pair of click conjugates comprising a first reporter portion and a second reactive functional group, wherein the second reactive functional group of the second member of the first pair of click conjugates is capable of reacting with the first reactive functional group of the first member of the pair of click conjugates. The second member of the pair of click conjugates can have a structure as provided in formula (IV). Finally, the signal from the first reporter moiety is detected.

[0361] refer to Figure 15 , the method for detecting one or more targets in a tissue sample using the click conjugates described herein can generally be divided into two stages. In the first stage, each target in the tissue sample is labeled with an enzyme (see box 155 and the steps contained therein). In the second stage, the reporter portion is deposited directly on or near each target (see box 165 and the steps contained therein), wherein the reporter portion is deposited using a pair of click conjugates listed herein (e.g., a first conjugate comprising a portion of a tissue-reactive portion and having a structure of any one of formulas (II) and (III) and a second conjugate comprising a reporter portion and having a structure of formula (IV)). The skilled artisan will understand that each of these general steps (step 170) can be repeated in multiplex assays to detect multiple different targets in a tissue sample. Each of these steps will be described in further detail herein.

[0362] In some embodiments, and before introducing any detection reagent, the tissue sample is pretreated with an enzyme inactivation composition to substantially or completely inactivate endogenous peroxidase activity. For example, in the case where cells or tissues contain endogenous peroxidase, the use of HRP-conjugated antibodies can result in high, non-specific background staining. This non-specific background can be reduced by pre-treating the sample with an enzyme inactivation composition as disclosed herein. In some embodiments, the sample is pre-treated with hydrogen peroxide (approximately 1% by weight to approximately 3% by weight of an appropriate pre-treatment solution) only to reduce endogenous peroxidase activity.

[0363] Reference again Figure 15 A tissue sample containing one or more targets is contacted with a first specific binding moiety specific for a first target to provide a first specific binding moiety-target complex (step 100). In some embodiments, the first specific binding moiety is an antibody or antibody conjugate (e.g., an unmodified antibody or an antibody conjugated to a detectable label, such as a hapten). In other embodiments, the first specific binding moiety is a nucleic acid probe conjugated to a detectable label, such as a hapten.

[0364] Subsequently, the first specific binding moiety-target complex is labeled with a first enzyme via the first specific binding moiety (step 110). In some embodiments, labeling of the target complex can be achieved using a secondary antibody, which is an anti-antibody antibody (e.g., an antibody specific for the first antibody, i.e., an anti-antibody antibody) or an anti-marker antibody (e.g., an anti-marker antibody or an anti-hapten antibody), which is conjugated to an enzyme (e.g., HRP, AP, etc.).

[0365] The tissue sample is then contacted with a first member of a first pair of click conjugates, wherein the first member of the first pair of click conjugates comprises a tissue-reactive portion and a first reactive functional group (step 120). The first member of the first pair of click conjugates can have a formula as provided in either formula (II) or (III). The first member of the first pair of click conjugates interacts / reacts with the first enzyme to form a reactive species or intermediate, wherein the reactive species or intermediate is capable of forming a covalent bond with the tissue sample directly on or near the first target, directly or indirectly. Next, a second member of the first pair of click conjugates is introduced (step 130), the second member of the first pair of click conjugates comprising a first reporter portion and a second reactive functional group, wherein the second reactive functional group of the second member of the first pair of click conjugates is capable of reacting with the first reactive functional group of the first member of the first pair of click conjugates. The second member of the first pair of click conjugates can have a structure as provided in formula (IV). Finally, a signal from the first reporter portion is detected (e.g., bright field microscopy) (step 140). In some embodiments, the first reporter portion is a chromophore. In some embodiments, the second member of the first pair of click conjugates is conjugated to at least two chromophores, and wherein the second member of the first pair of click conjugates has a structure of Formula (V).

[0366] The above process can be repeated for any number of targets in the sample (step 170). In some embodiments, an enzyme inactivation composition can be introduced to substantially or completely inactivate any enzyme from any upstream step. The tissue sample can then be contacted with a second specific binding moiety specific for a second target to provide a second specific binding moiety target complex (step 100). Subsequently, the second specific binding moiety-target complex is labeled with a second enzyme by the second specific binding moiety (step 110). The tissue sample is then contacted with a first member of a second pair of click conjugates, wherein the first member of the second pair of click conjugates comprises a quinone methide precursor or a tyramide moiety and a first reactive functional group (step 120). The first member of the second pair of click conjugates interacts with the second enzyme to form a reactive species, wherein the reactive species is capable of forming a covalent bond directly on or near the second target. The first member of the first pair of click conjugates can have a formula as provided in any one of formula (II) or (III). Next, a second member of a second pair of click conjugates is introduced (step 130), the second member of the second pair of click conjugates comprising a second reporter moiety and a second reactive functional group, wherein the second reactive functional group of the second member of the second pair of click conjugates is capable of reacting with the first reactive functional group of the first member of the second pair of click conjugates. The second member of the second pair of click conjugates can have a structure as provided in formula (IV). The second reporter moiety is then detected (step 140). This process can be repeated for a third, fourth, or nth target within the tissue sample (step 170).

[0367] The skilled person will understand that Figure 15 The steps described in can be performed sequentially (or continuously) or substantially simultaneously. For example, the tissue sample can be contacted with two specific binding moieties simultaneously at step 100 (wherein each specific binding moiety is specific for a particular target); and then each specific binding moiety-target complex is simultaneously labeled with a different enzyme at step 110. In these embodiments, any reagent used in steps 100 or 110 can be provided as a "pool" or "mixture" of reagents. Alternatively, a first specific binding moiety can be deposited (step 100) and the first specific binding moiety-target complex is subsequently labeled (step 110). Steps 100 and 110 can be repeated any number of times in succession before any click conjugates are introduced (step 150).

[0368] Subsequently, the tissue sample with the plurality of enzyme-labeled target complexes (steps 100, 110, and 150) can then be contacted with the plurality of click conjugates. The first members of the click conjugate pair can be added simultaneously at step 120, followed by the simultaneous introduction of the second members of the click conjugate pair at step 130. Alternatively, the first member of the first pair of click conjugates can be introduced, followed by the introduction of the second member of the first pair of click conjugates, and the sequential introduction of the first and second members of the click conjugate pair can be repeated any number of times (step 160) to introduce a reporter moiety for each labeled target complex.

[0369] Advantageously, for the methods just described, the first enzyme and the second enzyme are different enzymes. For example, the first enzyme can be a phosphatase or a phosphodiesterase, and the second enzyme can be a peroxidase. In certain embodiments, the first enzyme is alkaline phosphatase, and the second enzyme is horseradish peroxidase. Advantageously, the first enzyme does not interact with the first member of the second pair of click conjugates, so that the reactive intermediate derived from the first member of the second pair of click conjugates is deposited near the first target.

[0370] automation

[0371] The assays and methods disclosed herein can be automated and can be combined with sample processing equipment. The sample processing equipment can be automated equipment, such as the BENCHMARK XT instrument, SYMPHONY instrument, and BENCHMARK ULTRA instrument sold by Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. is the assignee of many U.S. patents that disclose systems and methods for performing automated analysis, including U.S. Patent Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, and U.S. Published Patent Application Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety. Alternatively, samples can be processed manually.

[0372] The sample processing device can apply a fixative to the sample. The fixative can include cross-linking agents (such as aldehydes, e.g., formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde cross-linking agents), oxidizing agents (e.g., 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., mercuric chloride, acetone, and picric acid), combination reagents (e.g., Carnoy's fixative, methacarn, Bouin's solution, B5 fixative, Rossmann's solution, and Gendel's solution), microwave and mixed fixatives (e.g., excluded volume fixation and vapor fixation).

[0373] If the sample is embedded in paraffin, the sample can be dewaxed using a sample processing device using an appropriate dewaxing fluid. After the waste removal agent removes the dewaxing fluid, any number of substances can be applied to the sample in succession. The substances can be used for pretreatment (e.g., protein-crosslinking, exposing nucleic acids, etc.), denaturation, hybridization, washing (e.g., stringent washing), detection (e.g., attaching visual or labeling molecules to probes), amplification (e.g., amplifying proteins, genes, etc.), counterstaining, coverslipping, etc.

[0374] The sample processing equipment can apply a wide range of substances to the sample. The substances include but are not limited to stains, probes, reagents, rinses and / or conditioners. The substances can be fluids (e.g., gases, liquids or gas / liquid mixtures) etc. The fluids can be solvents (e.g., polar solvents, non-polar solvents, etc.), solutions (e.g., aqueous solutions or other types of solutions) etc. Reagents can include but are not limited to stains, wetting agents, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.), antigen recovery fluids (e.g., antigen repair solutions based on aqueous or non-aqueous, antigen recovery buffers, etc.) etc. The probe can be an isolated nucleic acid or an isolated synthetic oligonucleotide attached to a detectable marker. The marker can include radioactive isotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent reagents, haptens and enzymes.

[0375] After processing the sample, the user can transport the slide carrying the sample to an imaging device. The imaging device used here is a bright field imager slide scanner. One bright field imager 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 (Patent Publication No. WO / 2011 / 049608) entitled IMAGING SYSTEM AND TECHNIQUES or in U.S. Patent Application Publication No. 2014 / 0178169, entitled IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME, filed on February 3, 2014. International Patent Application No. PCT / US2010 / 002772 and U.S. Patent Application Publication No. 2014 / 0178169 are incorporated by reference in their entireties. In other embodiments, the imaging device includes a digital camera coupled to a microscope.

[0376] Counterstaining

[0377] Counterstaining is a method in which a sample is post-processed after staining with a reagent to detect one or more targets so that their structure can be more easily observed under a microscope. For example, a counterstain is optionally used before a coverslip is applied to make immunohistochemical stains clearer. The color of the counterstain is different from the initial stain. Numerous counterstains are well known, such as hematoxylin, eosin, methyl green, methylene blue, Giemsa dye, alcian blue, and nuclear fast red. DAPI (4', 6-diamidino-2-phenylindole) is a fluorescent dye that can be used.

[0378] In some instances, more than one stain can be mixed together to create a counterstain. This provides flexibility and the ability to select stains. For example, a first stain can be selected for a mixture that has a specific property, but does not yet have a different desired property. A second stain can be added to the mixture that exhibits the desired property that is missing. For example, toluidine blue, DAPI, and pontamine sky blue can be mixed together to form a counterstain.

[0379] Detection and / or imaging

[0380] 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 certain embodiments, light microscopy is used for image analysis. Certain disclosed embodiments relate to acquiring digital images. This can be accomplished by combining a digital camera with a microscope. Image analysis software is used to analyze digital images obtained from stained samples. Color or fluorescence can be measured in several different ways. For example, color can be measured as red, blue, and green values; hue, saturation, and intensity values; and / or a spectral imaging camera can be used to measure specific wavelengths or wavelength ranges. The sample can also be assessed qualitatively and semi-quantitatively. Qualitative evaluation includes evaluating staining intensity, identifying positively stained cells and intracellular compartments involved in staining, and assessing overall sample or slide quality. Separate evaluations are performed on the test sample, and the analysis can include comparison with a known average value to determine whether the sample exhibits an abnormal state.

[0381] Reagent test kit

[0382] In some embodiments, the click conjugates can be used as part of a "detection kit". In some embodiments, the detection kit comprises at least a first click conjugate in a first container and a second click conjugate in a second container. The first click conjugate is the first member of a pair of click conjugates having a first reactive functional group; and the second click conjugate is the second member of a pair of click conjugates having a second reactive functional group, wherein the first and second reactive functional groups are capable of reacting with each other to form a covalent bond. In some embodiments, the first click conjugate is selected from a compound having a structure of either formula (II) or (III). In some embodiments, the second click conjugate is selected from a compound having a structure of formula (IV) or formula (V).

[0383] The detection kit may also include other reagents including a specific binding moiety and a secondary antibody specific for the specific binding moiety, the secondary antibody being conjugated to a detectable label. Of course, any kit may include other reagents, including buffers; counterstains; enzyme inactivation compositions; dewaxing solutions, etc., as needed for manual or automated target detection. The kit may also include instructions for using any components of the kit, including methods for applying the kit components to a tissue sample to detect one or more targets therein.

[0384] Samples and targets

[0385] The sample comprises biological components and is generally suspected of comprising one or more target molecules of interest. The target molecule can be located on the cell surface and the cell can be in suspension or in a tissue section. The target molecule can also be detected intracellularly and after cell lysis or cell penetration by a probe. Those of ordinary skill in the art will recognize that the method for detecting the target molecule in the sample will vary depending on the type of sample and probe used. Methods for collecting and preparing samples are known in the art.

[0386] In the embodiment of the method and with the sample such as tissue or other biological samples used together with the compositions disclosed herein can be prepared by those of ordinary skill using any method known in the art. Sample can be obtained from the subject for conventional screening or from the subject suspected of having a disease, such as genetic abnormality, infection or neoplasia. The described embodiment of the disclosed method can also be applied to samples that do not have genetic abnormality, disease, disease etc. that are referred to as "normal" samples. Such normal samples can be used as (among other things) for comparison with other samples. Sample can be analyzed for many different purposes. For example, sample can be used for scientific research or for diagnosing suspicious diseases, or as a prognostic indicator for successful treatment, survival etc.

[0387] The sample may include a variety of targets that can be specifically bound by a probe or reporter molecule. The target may be a nucleic acid sequence or a protein. When it comes to a target protein, it should be understood throughout this disclosure that a nucleic acid sequence associated with the protein may also be used as a target. In some instances, the target is a protein or nucleic acid molecule that is derived from a pathogen such as a virus, bacteria, or intracellular parasite, such as from a viral genome. For example, a target protein may be produced by a target nucleic acid sequence that is associated with a disease (e.g., associated with it, causally related, etc.).

[0388] The target nucleic acid sequence can vary substantially in size. Without limitation, the nucleic acid sequence can have a variable number of nucleic acid residues. For example, the target nucleic acid sequence can have at least about 10 nucleic acid residues, or at least about 20, 30, 50, 100, 150, 500, 1000 residues. Similarly, the target polypeptide can vary substantially in size. Without limitation, the target polypeptide will comprise at least one epitope that binds to a peptide-specific antibody or a fragment thereof. In some embodiments, the polypeptide can comprise at least two epitopes that bind to a peptide-specific antibody or a fragment thereof.

[0389] In a specific, non-limiting example, the target protein is produced by a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) associated with a neoplasm (e.g., a cancer). Many chromosomal abnormalities (including translocations and other rearrangements, amplifications, or deletions) have been identified in neoplastic cells, particularly in cancer cells such as B-cell and T-cell leukemias, lymphomas, breast cancer, colon cancer, neurological cancers, and the like. Thus, in some instances, at least a portion of the target molecule is produced by a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) that is amplified or deleted in at least one subset of cells in a sample.

[0390] Oncogenes are known to be the cause of several human malignancies. For example, chromosomal rearrangements involving the SYT gene located in the breakpoint region of chromosome 18q11.2 are common in synovial sarcoma soft tissue tumors. The t(18q11.2) translocation can be identified, for example, using probes with different markers: the first probe includes an FPC nucleic acid molecule generated from a target nucleic acid sequence extending distally from the SYT gene, and the second probe includes 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 with a deletion of t(18q11.2) in the SYT gene region show two fused (generated by two adjacent markers) signals, reflecting two intact copies of SYT. Abnormal cells with t(18q11.2) show a single fusion signal.

[0391] In other examples, the target protein produced by a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) is selected, and the nucleic acid sequence is a tumor suppressor gene that is deleted (lost) in malignant cells. For example, the p16 region (including D9S1749, D9S1747, p16 (INK4A), p14 (ARF), D9S1748, p15 (INK4B) and D9S1752) located in chromosome 9p21 is deleted in certain bladder cancers. Chromosomal deletions involving the distal region of the short arm of chromosome 1 (including, for example, SHGC57243, TP73, EGFL3, ABL2, ANGPTL1 and SHGC-1322) and the pericentromeric region (pericentromeric) of chromosome 19 (e.g., 19p13-19q13) (including, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2 and GLTSCR1) are characteristic molecular features of certain types of solid tumors of the central nervous system.

[0392] The aforementioned examples are provided for illustrative purposes only and are not intended to be limiting.Many other cytogenetic abnormalities associated with neoplastic transformation and / or growth are known to those of ordinary skill in the art. Target proteins produced by nucleic acid sequences (e.g., genomic target nucleic acid sequences) that have been associated with neoplastic transformation and can be used in the disclosed methods also include the EGFR gene (7p12; e.g., GENBANK™ Accession No. NC-000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21; e.g., GENBANK™ Accession No. NC-000008, nucleotides 128817498-128822856), D5S271 (5p15.2), the lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK™ Accession No. NC-000008, nucleotides 19841058-19869049), RB1 (13q14; e.g., GENBANK™ Accession No. NC-000013, nucleotides 47775912-47954023), p53 (8q24.21; e.g., GENBANK™ Accession No. NC-000008, nucleotides 128817498-128822856), (17p13.1; e.g., GENBANK™ Accession No. NC-000017, complementary sequence, nucleotides 7512464-7531642)), N-MYC (2p24; e.g., GENBANK™ Accession No. NC-000002, complementary sequence, nucleotides 151835231-151854620), CHOP (12q13; e.g., GENBANK™ Accession No. NC-000012, complementary sequence, nucleotides 56196638-56200567), FUS (16p11.2; e.g., GENBANK™ Accession No. NC-000016, complementary sequence, nucleotides 31098954-31110601), FKHR (13p14; e.g., GENBANK™ Accession No. NC-000013, complementary sequence, nucleotides 40027817-40138734), and, e.g., ALK (2p23; e.g., GENBANK™ Accession No. NC-000002, complementary sequence, nucleotides 29269144-29997936), Ig heavy chain; CCND1 (11q13; e.g., GENBANK™ Accession No. NC-000011, nucleotides 69165054.69178423), BCL2 (18q21.3; e.g., GENBANK™ Accession No. NC-000018, complementary sequence, nucleotides 58941559-59137593), BCL6 (3q27; e.g., GENBANK™ Accession No. NC-000003, complementary sequence, nucleotides 188921859-188946169), MALF1, AP1 (1p32-p31; e.g., GENBANK™ Accession No. NC-000001, complementary sequence, nucleotides 59019051-59022373), TOP2A (17q21-q22; e.g., GENBANK™ Accession No. NC-000017, complementary sequence, nucleotides 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK™ Accession No. NC-000001 e.g., GENBANK™ Accession No. NC-000021, complementary sequence, nucleotides 41758351-41801948), ERG (21q22.3; e.g., GENBANK™ Accession No. NC-000021, complementary sequence, nucleotides 38675671-38955488); ETV1 (7p21.3; e.g., GENBANK™ Accession No. NC-000007, complementary sequence, nucleotides 13897379-13995289), EWS (22q12.2; e.g., GENBANK™ Accession No. NC-000022, nucleotides 27994271-28026505); FLI1 (11q24.1-q24.3; e.g., GENBANK™ Accession No. NC-000011, nucleotides 128069199-128187521), PAX3 (2q35-q37; e.g., GENBANK™ Accession No. NC-000002, complementary sequence, nucleotides 222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK™ Accession No. NC-000001, nucleotides 18830087-18935219), PTEN (10q23.3; e.g., GENBANK™ Accession No. NC-000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g., GENBANK™ accession number NC-000019, complementary sequence, nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK™ Accession No. NC-000001, complementary sequence, nucleotides 40133685-40140274), REL (2p13-p12; e.g., GENBANK™ Accession No. NC-000002, nucleotides 60962256-61003682), and CSF1R (5q33-q35; e.g., GENBANK™ Accession No. NC-000005, complementary sequence, nucleotides 149413051-149473128).

[0393] In other examples, the target protein is selected from viruses or other microorganisms associated with a disease or condition. The detection of a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) of viral or microbial origin in a cell or tissue sample indicates the presence of an organism. For example, a target peptide, polypeptide, or protein 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, Entamoeba histolytica, and Giardia lamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesia species).

[0394] In some examples, the target protein is produced by a nucleic acid sequence from a viral genome (eg, a genomic target nucleic acid sequence). Exemplary viruses and corresponding genomic sequences (GENBANK™ RefSeq accession numbers are in parentheses) include human adenovirus A (NC_001460), human adenovirus B (NC_004001), human adenovirus C (NC_001405), human adenovirus D (NC_002067), human adenovirus E (NC_003266), human adenovirus F (NC_001454), human astrovirus (NC_001943), human BK polyomavirus (V01109; GI:60851), human bocavirus (NC_007455), human coronavirus 229E (NC_002645), human coronavirus HKU1 (NC_006577), human coronavirus NL63 (NC_005831), human coronavirus OC43 (NC_005147), human enterovirus A (NC_001612), human enterovirus B (NC_001613), human enterovirus C (NC_001614), human enterovirus E (NC_003266), human enterovirus F (NC_001454), human astrovirus (NC_001943), human BK polyomavirus (V01109; GI:60851), human bocavirus (NC_007455), human coronavirus 229E (NC_002645), human coronavirus HKU1 (NC_006577), human coronavirus NL63 (NC_005831), human coronavirus OC43 (NC_005147), human enterovirus (NC_001472), human enterovirus C (NC_001428), human enterovirus D (NC_001430), human erythrocytic virus V9 (NC_004295), human foamy virus (NC_001736), human herpesvirus 1 (herpes simplex virus type 1) (NC_001806), human herpesvirus 2 (herpes simplex virus type 2) (NC_001798), human herpesvirus 3 (varicella-zoster virus) (NC_001348), human herpesvirus 4 type 1 (Epstein-Barr virus type 1) (NC_007605), human herpesvirus 4 type 2 (Epstein-Barr virus type 2) (NC_009334), human herpesvirus 5 strain AD 169 (NC_001347), human herpesvirus 5 strain Merlin strain (NC_006273), human herpesvirus 6A (NC_001664), human herpesvirus 6B (NC_000898), human herpesvirus 7 (NC_001716), human herpesvirus 8 type M (NC_003409), human herpesvirus 8 type P (NC_009333), human immunodeficiency virus 1 (NC_001802), human immunodeficiency virus 2 (NC_001722), human metapneumovirus (NC_004148), human papillomavirus-1 (NC_001356), human papillomavirus-18 (NC_001357), human papillomavirus-2 (NC_001352), human papillomavirus-54 (NC_001676), human papillomavirus-61 (NC_001694), human papillomavirus-cand90 (NC_004104), human papillomavirus RTRX7 (NC_004761), human papillomavirus type 10(NC_001576), HPV 101 (NC_008189), HPV 103 (NC_008188), HPV 107 (NC_009239), HPV 16 (NC_001526), ​​HPV 24 (NC_001683), HPV 26 (NC_001583), HPV 32 (NC_001586), HPV 34 (NC_001587), HPV 4 (NC_001457), HPV 41 (NC_001354), HPV 48 (NC_001690), HPV 49 (NC_001591), HPV 5 (NC_001531), human papillomavirus type 50 (NC_001691), human papillomavirus type 53 (NC_001593), human papillomavirus type 60 (NC_001693), human papillomavirus type 63 (NC_001458), human papillomavirus type 6b (NC_001355), human papillomavirus type 7 (NC_001595), human papillomavirus type 71 (NC_002644), human papillomavirus type 9 (NC_001596), human papillomavirus type 92 (NC_004500), human papillomavirus type 96 (NC_005134), human parainfluenza virus 1 (NC_003461), human parainfluenza virus 2 (NC_003443), human parainfluenza virus 3 (NC_001796), human double echovirus (NC_001897), human parvovirus 4 (NC_007018), human parvovirus B19 (NC_000883), human respiratory syncytial virus (NC_001781), human rhinovirus A (NC_001617), human rhinovirus B (NC_001490), human foamy retrovirus (NC_001795), human T-lymphotropic virus 1 (NC_001436), human T-lymphotropic virus 2 (NC_001488).

[0395] In some instances, the target protein is produced by a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) from an oncogenic virus such as Epstein-Barr virus (EBV) or human papillomavirus (HPV, e.g., HPV16, HPV18). In other instances, the target protein produced by a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) is from a pathogenic virus 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). Example

[0396] The non-limiting examples presented herein each incorporate the use of at least one pair of click conjugates. Applicants believe that the click conjugates disclosed herein are suitable for use in IHC assays, including multiplexed IHC assays and ISH assays, as demonstrated in the following examples.

[0397] General Immunohistochemistry (IHC) Protocol

[0398] Unless otherwise specified, all IHC staining experiments were performed on the VENTANA BenchMark ® The staining was performed on the XT automated tissue staining platform, and the reagents used in these protocols were from Ventana Medical Systems, Inc. (Tucson, AZ, USA; “Ventana”). Polyclonal goat anti-rabbit antibody, polyclonal goat anti-mouse antibody, horseradish peroxidase (HRP), and alkaline phosphatase (AP) were obtained from Roche Diagnostics (Mannheim, Germany).

[0399] The following general steps were performed: (1) deparaffinization with EZ Prep detergent solution (Ventana Medical Systems, Inc. (VMSI), #950-101) (75°C; 20 minutes); (2) washing with reaction buffer (VMSI, #950-300); (3) antigen retrieval in cell conditioning 1 (VMSI #950-124) (100°C; time depends on the target antigen); (4) washing (same as step 2); (5) for protocols with a subsequent HRP detection step, use iVIEW inhibitor (VMSI, E253-2187) to inactivate endogenous peroxidase (37°C; 4 minutes); (6) wash (same as step 2); (7) primary antibody incubation (anti-target antibody) at 37°C for a time that depends on the primary antibody and ranges from 8 to 32 minutes; (8) wash (same as step 2); and (9) secondary antibody incubation with a goat polyclonal anti-species antibody conjugated to an enzyme (HRP or AP, 37°C; 8 to 12 minutes). All subsequent reagent incubation steps were separated by washes as in step (2). Targets were detected as described in Examples 1-6.

[0400] Example 1: "Click" amplification using compounds of formula (II)

[0401] Three examples of IHC "click" amplification using different compounds of formula (II) are illustrated in Figure 7A 、 7B and 7C. In general, each IHC assay was performed according to the methods disclosed herein. Figure 7A、 7B In 7C and 7D, each tissue sample is first contacted with a primary antibody specific for a particular target ( Figure 7A , CD8; Figure 7B , Bcl6; and Figure 7C , Ki67). After the introduction of the corresponding primary antibody, each antibody-target complex is labeled with an enzyme, such as by introducing a secondary antibody coupled to alkaline phosphatase (AP) enzyme (e.g., goat anti-rabbit antibody-AP conjugate or goat anti-mouse antibody-AP conjugate).

[0402] Next, the first member of a pair of click conjugates is introduced and reacted with each AP-labeled target. Figure 7A In the present invention, a compound of formula (II) comprising a quinone methide precursor linked to a DBCO-reactive functional group is introduced and reacts with target-bound alkaline phosphatase to form a quinone methide-DBCO tissue conjugate complex. Subsequently, a conjugate of formula (IV) comprising the chromogen TAMRA and an azide-reactive functional group is introduced and reacts with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 7A Staining for CD8 glycoprotein is clearly shown within the tonsil tissue sample.

[0403] exist Figure 7B In the present invention, a compound of formula (II) comprising a quinone methide precursor linked to an azide-reactive functional group is introduced and reacts with target-bound alkaline phosphatase to form a quinone methide-azide tissue conjugate complex. Subsequently, a conjugate of formula (IV) comprising the chromogen TAMRA and a DBCO-reactive functional group is introduced and reacts with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 7B Staining for B-cell lymphoma 6 protein is clearly shown within the tonsil tissue sample.

[0404] exist Figure 7C In the present invention, a compound of formula (II) comprising a quinone methide precursor linked to a TCO-reactive functional group is introduced and reacts with target-bound alkaline phosphatase to form a quinone methide-TCO tissue conjugate complex. Subsequently, a conjugate of formula (IV) comprising the chromogen TAMRA and a tetrazine-reactive functional group is introduced and reacts with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 7C The staining of Ki67 protein in the tonsil tissue sample is clearly shown.

[0405] Example 2: "Click" amplification using compounds of formula (III)

[0406] Three examples of IHC "click" amplification using different compounds of formula (III) are illustrated in Figure 8A 、 8Band 8C. In general, each IHC assay was performed according to the methods disclosed herein. Figure 8A 、 8B In 8C and 8D, each tissue sample is first contacted with a primary antibody specific for a particular target ( Figure 8A , CD8; Figure 8B , Bcl6; and Figure 8C After the introduction of the corresponding primary antibody, each antibody-target complex is enzymatically labeled, such as by introducing a secondary antibody coupled to horseradish peroxidase (HRP) enzyme (e.g., goat anti-rabbit antibody-HRP conjugate or goat anti-mouse antibody-HRP conjugate).

[0407] Next, the first member of a pair of click conjugates is introduced and reacted with each HRP-labeled target. Figure 8A In the present invention, a compound of formula (III) comprising a tyramide linked to an azide-reactive functional group is introduced and reacts with target-bound HRP to form a tyramide-azide tissue conjugate complex. Subsequently, a compound of formula (IV) comprising the chromogen TAMRA and a DBCO-reactive functional group is introduced and reacts with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 8A Staining for CD8 glycoprotein is clearly shown within the tonsil tissue sample.

[0408] exist Figure 8B In the present invention, a compound of formula (III) comprising a tyramide linked to a DBCO-reactive functional group is introduced and reacts with target-bound HRP to form a tyramide-DBCO tissue conjugate complex. Subsequently, a compound of formula (IV) comprising a chromogen TAMRA and an azide-reactive functional group is introduced and reacts with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 8B The staining of Ki67 protein in the tonsil tissue sample is clearly shown.

[0409] exist Figure 8C In the present invention, a compound of formula (III) comprising a tyramide linked to a TCO-reactive functional group is introduced and reacts with target-bound HRP to form a tyramide-TCO tissue conjugate complex. Subsequently, a compound of formula (IV) comprising a chromogen TAMRA and a tetrazine-reactive functional group is introduced and reacts with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 8C Staining for CD8 glycoprotein is clearly shown within the tonsil tissue sample.

[0410] Example 3

[0411] Figure 9A, 9B, 9C, and 9D illustrate results from four different IHC assays. Each assay was performed using the general procedure described herein and exemplified in Example 1. As used herein, each assay used the same click conjugate of formula (II), i.e., a conjugate comprising a tyramide moiety conjugated to a DBCO-reactive functional group ("tyramide-DBCO"). However, four different click conjugates of formula (IV) were used for coupling to tyramide-DBCO, each having a different chromogen or color development system coupled to an azide-reactive functional group. Figure 9 As depicted in Figure A, the tyramide-DBCO conjugate is reacted with a click conjugate of formula (IV), wherein the click conjugate of formula (IV) comprises a coupled Cy5 chromogen. Figure 9 As depicted in B, the tyramide-DBCO conjugate is reacted with a click conjugate of formula (IV), wherein the click conjugate of formula (IV) comprises a coupled Dabsyl chromogen. Figure 9 As depicted in C, the tyramide-DBCO conjugate is reacted with a click conjugate of formula (IV), wherein the click conjugate of formula (IV) comprises both a TAMRA chromogen and a Dabcyl chromogen, wherein the two chromogens are coupled via a lysine scaffold. Figure 9 As depicted in D, the tyramide-DBCO conjugate is reacted with a click conjugate of formula (IV), wherein the click conjugate of formula (IV) comprises a coupled TAMRA chromogen. Thus, Figure 9 A to 9D each illustrate that a click conjugate species comprising a tissue-reactive promoiety and a specific reactive functional group can react with different compounds of formula (IV) having different chromogens to stain tissues in different colors.

[0412] Example 4: Comparison of “Traditional” TSA and “Click” Amplification in IHC Assays

[0413] Figure 10 Staining with a DAB control, various TSA chromogens (TSA-TAMRA, TSA-Cy5, and TSA-Dabsyl), and the disclosed TSA "click" conjugates (Tyramide-DBCO:TAMRA-Azide; Tyramide-DBCO:Cy5-Azide; and Tyramide-DBCO:Dabsyl-Azide) is comparatively illustrated.

[0414] Tissue samples labeled "DAB control" were stained in an IHC assay using a primary antibody specific for Ki67 and a goat anti-rabbit antibody conjugated to HRP. Following the addition of hydrogen peroxide and 3,3'-diaminobenzidine (DAB), the antigen was visualized via a brown precipitate produced by HRP. The DAB color tone was adjusted by the addition of copper sulfate.

[0415] Figure 10The tissue samples identified as those stained with TSA-TAMRA, TSA-Cy5 and TSA-Dabsyl were dyed in the IHC assay using traditional tyramide signal amplification technology. First, a primary antibody specific to Ki67 was introduced to form an anti-Ki67 complex. The primary antibody-Ki67 complex was then labeled with horseradish peroxidase by a secondary antibody, i.e., goat anti-rabbit antibody-HRP conjugate. Subsequently, each was independently introduced with tyramide coupled to a chromogen, i.e., TSA-TAMRA, TSA-Cy5 and TSA-Dabsyl, and each was subsequently deposited on or near the target after reacting with horseradish peroxidase.

[0416] Figure 10 Tissue samples identified as stained with TSA-DBCO:TAMRA-azide, Tyramide-DBCO:Cy5-azide; and Tyramide-DBCO:Dabsyl-azide were stained in IHC assays using the general techniques described herein and those provided in Example 2.

[0417] Tissues stained with Cy5 or Dabsyl in "click" amplification according to the methods described herein showed a significant increase in staining intensity compared to samples stained in conventional TSA assays, as shown in Figure 2. Figure 10 Clearly shown in.

[0418] Example 5: Comparison of “Traditional” TSA and “Click” Amplification in ISH Assays

[0419] Figure 11 Staining with various TSA chromogens (TSA-TAMRA, TSA-Cy5, and TSA-Dabsyl) and the disclosed TSA "click" conjugates (Tyramide-DBCO:TAMRA-Azide; Tyramide-DBCO:Cy5-Azide; and Tyramide-DBCO:Dabsyl-Azide) is comparatively illustrated.

[0420] Figure 11 The tissue sample that is identified as dyeing with TSA-TAMRA, TSA-Cy5 and TSA-Dabsyl is dyed in the ISH mensuration using traditional tyramide signal amplification technology.First, the nucleic acid probe specific for Her2 is introduced into the tissue sample, and the Her2 probe is put together with a detectable marker (i.e., DNP hapten). DNP is combined with rabbit anti-DNP antibody, and the rabbit anti-DNP antibody is then labeled with the goat anti-rabbit antibody that is put together to HRP. Subsequently, TSA-TAMRA, TSA-Cy5 and TSA-Dabsyl are introduced independently of one another, and are subsequently deposited on or near the target after reacting with horseradish peroxidase.

[0421] Figure 11Tissue samples identified as stained with TSA-DBCO:TAMRA-azide, tyramide-DBCO:Cy5-azide; and tyramide-DBCO:Dabsyl-azide were stained according to general techniques described herein (see, e.g., Figure 15 ).

[0422] Tissues stained with Cy5 or Dabsyl in "click" amplification according to the methods described herein showed a significant increase in staining intensity compared to samples stained in conventional TSA assays, as shown in Figure 2. Figure 11 Clearly shown in.

[0423] Example 6

[0424] Figure 12 Illustration of the difference in staining using a click conjugate of formula (IV) comprising a single reporter moiety compared to another click conjugate of formula (IV) comprising multiple reporter moieties. The tissue sample on the left was stained using a click conjugate of formula (IV) comprising a single TAMRA chromogen. The tissue sample on the right was stained using a click conjugate of formula (V) comprising at least two TAMRA chromogens coupled together using dendrimers.

[0425] Example 7

[0426] Figure 13 The staining of tissues with enzyme-tissue click adducts is described. IHC assays are performed according to the methods disclosed herein. After the introduction of rabbit anti-Ki67 primary antibody, each antibody-target complex is labeled with a goat anti-rabbit secondary antibody coupled to horseradish peroxidase (HRP) enzyme. Next, a compound of formula (III) comprising a tyramide connected to a DBCO reactive functional group (the first member of a pair of click conjugates) is introduced together with hydrogen peroxide and reacted with each HRP-labeled target. After the HRP reaction bound to the target, a tyramide-DBCO tissue conjugate complex is formed. Subsequently, a compound of formula (IV) comprising an enzyme AP and an azide reactive functional group (the second member of a pair of click conjugates) is introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct. The AP-tissue click adduct is then detected using QMSA-TAMRA colorimetric detection. Figure 13 An increase in Ki67 protein staining in tonsil tissue samples was clearly shown, corresponding to the increased concentration of AP-azide.

[0427] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the embodiments may be modified, if necessary, to employ concepts of the various patents, applications, and publications to provide additional embodiments.

[0428] Although the disclosure herein has been 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 many modifications may be made to the illustrative embodiments, and other arrangements may be designed without departing from the spirit and scope of the disclosure as defined by the appended claims.

Claims

1. A conjugate having formula (IIa): in A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine and hydroxylamine; A "linker" has formula (Ia): in d and e are each independently an integer ranging from 2 to 20; t and u are independently 0 or 1; Q is oxygen; R a and R b Each is H; and X and Y are independently a carbonyl group; R 1 is a phosphate group; R 2 It is a halogen; R 3 、R 5 and R 6 independently selected from hydrogen or an aliphatic group having 1 to 4 carbon atoms; R 4 is hydrogen or an aliphatic group having 1 to 4 carbon atoms; and R 7 is -C(O)N(H)(CH2) w NH-, wherein w is an integer ranging from 1 to 12.

2. The conjugate of claim 1, wherein R 6 、R 5 、R 4 and R 3 Each is hydrogen.

3. The conjugate of claim 1 or 2, wherein R 2 It's fluorine.

4. The conjugate of claim 1, wherein R 1 is phosphoric acid; R 2 is fluorine; and R 6 、R 5 、R 4 and R 3 Each is hydrogen.

5. The conjugate of claim 1 or 2, wherein R 1 is phosphoric acid and R 7 is -C(O)N(H)(CH2) w NH-, and w ranges from 2 to 10.

6. The conjugate of claim 5, wherein R 2 is fluorine; and R 6 、R 5 、R 4 and R 3 Each is hydrogen.

7. The conjugate of claim 6, wherein the "linker" comprises a PEG group.

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