Peptide nucleic acid conjugates

Abstract

The present disclosure relates to conjugates of specific binders and oligomers, i.e., [specific binder]-[oligomer] n (n is an integer ranging from 1 to 12), wherein the oligomer, in some embodiments, comprises a PNA sequence having at least one substituent at the gamma carbon. In some embodiments, the substituent at the gamma carbon, e.g., an amino acid, peptide, miniPEG, or polymer, comprises at least one reporter moiety. [Selected Figure] Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of International Application No. PCT / US2017 / 66976, filed December 18, 2017, the disclosure of which is incorporated herein by reference in its entirety, and also of the filing date of U.S. Provisional Patent Application No. 62 / 599,810, filed December 18, 2017. [Background technology]

[0002] Cell staining methods, including immunohistochemistry (IHC) and in situ hybridization analysis (ISH), are useful tools in histological diagnosis and tissue morphology research. IHC uses specific binding agents or moieties, such as antibodies, to detect antigens of interest that may be present in tissue samples. IHC is widely used in clinical and diagnostic applications, such as diagnosing specific disease states or conditions. For example, specific cancer types can be diagnosed based on the presence of specific marker molecules in samples obtained from a subject. IHC is also widely used in basic research to understand the distribution and localization of biomarkers within various tissues. Biological samples can also be examined using in situ hybridization techniques, such as silver in situ hybridization (SISH), chromogenic in situ hybridization (CISH), and fluorescent in situ hybridization (FISH), collectively referred to as ISH. ISH differs from IHC in that it detects nucleic acids in tissues, whereas IHC detects proteins in tissues.

[0003] Characterization and quantification of the large number of proteins expressed by an organism's genome is the focus of proteomics. Multiplex immunohistochemistry (mIHC) represents a significant unmet technical need for the detection and analysis of multivariate protein targets in paraffin-embedded, formalin-fixed tissues with broad application in research and diagnostics. Multiplex immunohistochemistry (mIHC) techniques attempt to address the need for the detection and analysis of multivariate protein targets in formalin-fixed, paraffin-embedded (FFPE) tissues. Effective mIHC techniques have broad application in research and diagnostics. However, few, if any, efficient and reproducible methods exist that enable simultaneous and quantitative detection of multiple protein targets in tissues.

[0004] A key constraint of translational research in clinical trial settings is the often limited amount of tissue available for biomarker analysis. Furthermore, this tissue is frequently archived and stored in FFPE blocks. Traditional methods of gene expression analysis have limited clinical applicability. For example, RT-PCR measures the expression of a single gene at a time, while multiplexed expression profiling techniques, such as microarrays, which cover thousands of transcripts, are often expensive and lack flexibility and reproducibility when evaluating low-quality RNA samples, such as those derived from FFPE samples. Evaluation of these assays is semi-quantitative and inherently subjective. For this reason, there has been increasing interest in developing quantitative, highly multiplexed assays that enable profiling of multiple markers in a single assay. Therefore, platforms that enable multiplexed analysis of biomarkers from limited amounts of poor-quality material are highly attractive.

[0005] The NanoString nCounter technology, which allows for the direct, automated detection of nucleic acids (DNA and / or RNA), is a relatively new technique that has been adopted for a variety of clinical and research applications. Typically, the target nucleic acid (DNA or RNA) is hybridized to a biotinylated DNA strand (capture strand), allowing for immobilization of the nucleic acid to the streptavidin surface inside the nCounter cartridge and a fluorescently labeled DNA strand (reporter strand, 7 kb, of which approximately 50 bases are complementary to the target nucleic acid). Automated digital readout of the fluorescently labeled reporter strand is believed to enable non-amplified measurement of up to 800 nucleic acid targets within a single sample.

[0006] DNA antibody barcoding consists of labeling an antibody with a cleavable DNA strand that can be used as a unique molecular tag. By combining DNA antibody barcoding with the NanoString nCounter technology, we have extended the NanoString nCounter technology to encompass applications involving multiplexed protein analysis.

[0007] Synthetic molecules capable of binding with high affinity and sequence specificity to selected targets in genetic sequences are of interest in medical and biotechnology settings. They hold promise as molecular tools for the development of gene therapy agents, diagnostic devices for genetic analysis, and nucleic acid manipulation. Peptide nucleic acids (PNAs) are nucleic acid analogs in which the sugar phosphate backbone of natural nucleic acids is replaced by a synthetic peptide backbone, typically formed from N-(2-amino-ethyl)-glycine units, resulting in achiral and uncharged mimics. They are chemically stable and resistant to hydrolytic (enzymatic) cleavage, and are therefore expected to survive degradation within living cells. PNAs are capable of sequence-specific recognition of DNA and RNA according to the Watson-Crick hydrogen bonding scheme, and hybrid complexes exhibit exceptional thermal stability and unique ionic strength effects. Because PNAs contain no charge, PNA-DNA hybridization is stronger than DNA-DNA hybridization for the same sequence. Summary of the Invention

[0008] One aspect of the present disclosure is a conjugate having the structure of formula (IA): TIFF0007727385000001.tif25170[In the formula, The "specific binding entity" is selected from the group consisting of an antibody (e.g., a primary or secondary antibody), an antibody fragment, a drug / antibody conjugate, and a nucleic acid; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; Z is selected from the group consisting of a PNA sequence comprising at least one nucleotide having a substitution at the gamma carbon position, an uncharged DNA sequence, and a DNA sequence comprising charged and uncharged bases; X is a label; Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms; m is 0 or an integer ranging from 1 to 6; z is 0 or 1; n is an integer ranging from 1 to 12].

[0009] In some embodiments, X is biotin, an enzyme, a chromogen, a fluorophore, a hapten, or a mass spectrometry tag.

[0010] In some embodiments, the "specific binder" is a primary antibody. In some embodiments, the "specific binder" is a secondary antibody. In some embodiments, the "specific binder" is a nucleic acid (e.g., DNA or RNA).

[0011] In some embodiments, Z comprises a DNA sequence that contains only uncharged DNA bases. In some embodiments, Z comprises a DNA sequence that contains a mixture of charged and uncharged bases. In some embodiments, Z comprises a DNA sequence in which at least 50% of the bases in the DNA sequence are uncharged.

[0012] In some embodiments, Z comprises a PNA sequence having at least two PNA bases with substituents at the gamma positions. In some embodiments, Z comprises a PNA sequence having at least three PNA bases with substituents at the gamma positions. In some embodiments, Z comprises a PNA sequence having at least four PNA bases with substituents at the gamma positions.

[0013] In some embodiments, at least one substituted nucleotide of the PNA sequence comprises a lysine residue or a lysine residue containing a reporter moiety. In some embodiments, at least one substituted nucleotide of the PNA sequence comprises a miniPEG containing at least one reporter moiety. In some embodiments, at least one substituted nucleotide of the PNA sequence comprises a polymer having at least one reporter moiety. In some embodiments, at least one substituted nucleotide of the PNA sequence comprises a peptide containing at least one reporter moiety (e.g., a chromogen, a fluorophore).

[0014] In some embodiments, the PNA sequence comprises about 5 to 20 bases. In some embodiments, the PNA sequence comprises about 5 to 15 bases. In some embodiments, the PNA sequence comprises about 5 to 10 bases. In some embodiments, the PNA sequence comprises about 15 bases. In some embodiments, the PNA sequence comprises about 10 bases.

[0015] In some embodiments, the "specific binder" is a primary antibody and Z is a PNA sequence or a PNA sequence having one or more substituents at the gamma position. In some embodiments, the "specific binder" is a primary antibody and Z is a PNA sequence having 10 nucleotides. In some embodiments, the "specific binder" is a primary antibody and Z is a PNA sequence having one or more substituents at the gamma position.

[0016] In some embodiments, a "linker" comprises a cleavable group, e.g., a photocleavable group, an enzymatically cleavable group, a chemically cleavable group, a group cleavable at a particular pH, etc. In some embodiments, a "linker" comprises one or more groups that confer water solubility as disclosed herein (e.g., one or more PEG groups).

[0017] Another aspect of the present disclosure is a conjugate having the structure of formula (IIB): TIFF0007727385000002.tif25170[In the formula, The "specific binding entity" is selected from the group consisting of an antibody, an antibody fragment, a drug / antibody conjugate, and a nucleic acid; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; A "PNA" is a PNA sequence containing at least one nucleotide with a substitution at the gamma carbon position; X is a label; Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms; m is 0 or an integer ranging from 1 to 6; z is 0 or 1; n is an integer ranging from 1 to 12].

[0018] In some embodiments, X is biotin, an enzyme, a chromogen, a fluorophore, a hapten, and a mass spectrometry tag.

[0019] In some embodiments, the PNA sequence has at least two gamma PNA bases. In some embodiments, each of the at least two gamma PNA bases is substituted with the same amino acid, miniPEG, peptide, or polymer. In some embodiments, each of the at least two gamma PNA bases is substituted with a different amino acid, miniPEG, peptide, or polymer. In some embodiments, the amino acid, miniPEG, peptide, or polymer comprises at least one reporter moiety (e.g., fluorophore, chromogen). In some embodiments, the PNA sequence has at least three gamma PNA bases. In some embodiments, the PNA sequence has at least four gamma PNA bases.

[0020] In some embodiments, the PNA sequence comprises about 5 to 20 bases. In some embodiments, the PNA sequence comprises about 5 to 15 bases. In some embodiments, the PNA sequence comprises about 5 to 10 bases. In some embodiments, the PNA sequence comprises about 15 bases. In some embodiments, the PNA sequence comprises about 10 bases.

[0021] In some embodiments, the "specific binder" is a primary antibody. In some embodiments, the "specific binder" is a secondary antibody. In some embodiments, X is biotin. In some embodiments, m is 0, z is 0, and n is greater than 1. In some embodiments, n is an integer ranging from 2 to 6. In some embodiments, the "linker" comprises at least one PEG group.

[0022] In some embodiments, the "specific binder" is a primary antibody and the PNA sequence comprises 10 nucleotides. In some embodiments, the "specific binder" is a primary antibody and the PNA sequence comprises at least two nucleotides with substitutions at gamma positions.

[0023] In some embodiments, the "linker" has the structure shown in formula (IVA): TIFF0007727385000003.tif40170[In the formula, d and e are each independently an integer ranging from 2 to 20; Q is a bond, O, S, or N(R c )(R d ) and; R a and R b are independently H, a C1-C4 alkyl group, F, Cl, or N(R c )(R d ) and; R c and R d are independently CH or H; A and B are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having from 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.

[0024] In some embodiments, d and e are integers ranging from 2 to 6. In some embodiments, at least one of A or B comprises a cleavable moiety. In some embodiments, the cleavable moiety is a photocleavable group. In some embodiments, the cleavable moiety is a chemically cleavable group. In some embodiments, the "specific binder" is an antibody, the "linker" comprises at least one PEG group, m is 0, z is 0, and n is greater than 1. In some embodiments, the "specific binder" is an antibody, the "linker" comprises at least one PEG group, and n is greater than 1. In some embodiments, the "linker" further comprises at least one cleavable group. In some embodiments, X is a hapten.

[0025] Another aspect of the present disclosure is an oligomer having the structure of formula (III): TIFF0007727385000004.tif21170[In the formula, T is a group having 1 to 4 carbon atoms, optionally substituted with O, N, or S, and having a terminal reactive moiety; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; A "PNA" is a PNA sequence containing at least one nucleotide with a substitution at the gamma carbon position; X is a label; Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms; m is 0 or an integer ranging from 1 to 6; z is 0 or 1].

[0026] In some embodiments, X is biotin, an enzyme, a chromogen, a fluorophore, a hapten, and a mass spectrometry tag.

[0027] In some embodiments, X is biotin.

[0028] In some embodiments, the PNA sequence has at least two gamma PNA bases.

[0029] In some embodiments, each of at least two gamma PNA bases is substituted with the same amino acid, miniPEG, peptide, or polymer. In some embodiments, each of at least two gamma PNA bases is substituted with a different amino acid, miniPEG, peptide, or polymer. In some embodiments, the amino acid, miniPEG, peptide, or polymer comprises at least one reporter moiety (e.g., fluorophore, chromogen). In some embodiments, the PNA sequence has at least three gamma PNA bases. In some embodiments, the PNA sequence has at least four gamma PNA bases.

[0030] In some embodiments, at least one substituted nucleotide of the PNA sequence comprises a lysine residue, a peptide, or a miniPEG, each of which may have one or more reporter moieties linked thereto.

[0031] In some embodiments, PNA sequences, including PNA sequences with one or more substituents at gamma positions, comprise about 5 to 20 bases. In some embodiments, PNA sequences, including PNA sequences with one or more substituents at gamma positions, comprise about 5 to 15 bases. In some embodiments, PNA sequences, including PNA sequences with one or more substituents at gamma positions, comprise about 5 to 10 bases. In some embodiments, PNA sequences, including PNA sequences with one or more substituents at gamma positions, comprise about 15 bases. In some embodiments, PNA sequences, including PNA sequences with one or more substituents at gamma positions, comprise about 10 bases.

[0032] In some embodiments, the "specific binder" is a primary antibody and the PNA sequence comprises 10 nucleotides. In some embodiments, the "specific binder" is a primary antibody and the PNA sequence comprises at least one substituent at one gamma position.

[0033] In some embodiments, m is 0, z is 0, and n is greater than 1.

[0034] In some embodiments, n is an integer ranging from 2 to 6. In some embodiments, the "linker" comprises at least one PEG group. In some embodiments, the "linker" has the structure shown in formula (IVA): TIFF0007727385000005.tif40170[In the formula, d and e are each independently an integer ranging from 2 to 20; Q is a bond, O, S, or N(R c )(R d ) and; R a and Rb are independently H, a C1-C4 alkyl group, F, Cl, or N(R c )(R d ) and; R c and R d are independently CH or H; A and B are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having from 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.

[0035] In some embodiments, d and e are integers ranging from 2 to 6. In some embodiments, at least one of A or B comprises a cleavable moiety. In some embodiments, the cleavable moiety is a photocleavable group. In some embodiments, the cleavable moiety is a chemically cleavable group.

[0036] Another aspect of the present disclosure is a method for detecting a target in a sample, comprising: (a) contacting the sample with a first conjugate, wherein the first conjugate has formula (IA): TIFF0007727385000006.tif33170[In the formula, The "specific binding entity" is selected from the group consisting of an antibody, an antibody fragment, a drug / antibody conjugate, and a nucleic acid; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; Z is selected from the group consisting of a PNA sequence comprising at least one nucleotide having a substitution at the gamma carbon position, an uncharged DNA sequence, and a DNA sequence comprising charged and uncharged bases; X is a label; Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms; m is 0 or an integer ranging from 1 to 6; z is 0 or 1; n is an integer ranging from 1 to 12. and contacting the compound having the structure: (b) contacting the sample with a first detection reagent to facilitate detection of the first conjugate; and

[0037] In some embodiments, the method includes contacting the sample with a second conjugate of formula (IA), wherein the first and second conjugates are different (e.g., comprise different specific binders and / or different labels).

[0038] In some embodiments, X is selected from the group consisting of biotin, an enzyme, a chromogen, a fluorophore, a hapten, and a mass spectrometry tag.

[0039] In some embodiments, the "specific binder" is a primary antibody and Z is a PNA sequence or a gamma PNA sequence. In some embodiments, the "specific binder" is a primary antibody and Z is a PNA sequence having 10 nucleotides. In some embodiments, the "specific binder" is a primary antibody and Z is a gamma PNA sequence.

[0040] In some embodiments, Z comprises a DNA sequence that contains only uncharged DNA bases. In some embodiments, Z comprises a DNA sequence that contains a mixture of charged and uncharged bases. In some embodiments, Z comprises a DNA sequence in which at least 50% of the bases in the DNA sequence are uncharged.

[0041] In some embodiments, the PNA sequence has at least two gamma PNA bases. In some embodiments, each of the at least two gamma PNA bases is substituted with the same amino acid, miniPEG, peptide, or polymer. In some embodiments, each of the at least two gamma PNA bases is substituted with a different amino acid, miniPEG, peptide, or polymer. In some embodiments, the amino acid, miniPEG, peptide, or polymer comprises at least one reporter moiety (e.g., fluorophore, chromogen). In some embodiments, the PNA sequence has at least three gamma PNA bases. In some embodiments, the PNA sequence has at least four gamma PNA bases.

[0042] In some embodiments, the PNA sequence comprises about 5 to 20 bases. In some embodiments, the PNA sequence comprises about 5 to 15 bases. In some embodiments, the PNA sequence comprises about 5 to 10 bases. In some embodiments, the PNA sequence comprises about 15 bases. In some embodiments, the PNA sequence comprises about 10 bases.

[0043] Another aspect of the present disclosure is a method for detecting a target in a sample, comprising: (a) contacting the sample with a first PNA conjugate, wherein the first PNA conjugate has formula (IIB): TIFF0007727385000007.tif30170[In the formula, The "specific binding entity" is selected from the group consisting of an antibody, an antibody fragment, a drug / antibody conjugate, and a nucleic acid; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; A "PNA" is a PNA sequence containing at least one nucleotide with a substitution at the gamma carbon position; X is a label; Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms; m is 0 or an integer ranging from 1 to 6; z is 0 or 1; n is an integer ranging from 1 to 12. and contacting the compound having the structure: (b) contacting the sample with a first detection reagent to facilitate detection of the PNA conjugate; and

[0044] In some embodiments, the method includes contacting the sample with a second PNA conjugate (e.g., another conjugate of formula (IIB)), where the first PNA conjugate and the second PNA conjugate are different, i.e., have at least one component or moiety that is different.

[0045] In some embodiments, X is selected from the group consisting of biotin, an enzyme, a chromogen, a fluorophore, a hapten, and a mass spectrometry tag.

[0046] In some embodiments, the PNA sequence has at least two gamma PNA bases. In some embodiments, each of the at least two gamma PNA bases is substituted with the same amino acid, miniPEG, peptide, or polymer. In some embodiments, each of the at least two gamma PNA bases is substituted with a different amino acid, miniPEG, peptide, or polymer. In some embodiments, the amino acid, miniPEG, peptide, or polymer comprises at least one reporter moiety (e.g., fluorophore, chromogen). In some embodiments, the PNA sequence has at least three gamma PNA bases. In some embodiments, the PNA sequence has at least four gamma PNA bases.

[0047] In some embodiments, the PNA sequence comprises about 5 to 20 bases. In some embodiments, the PNA sequence comprises about 5 to 15 bases. In some embodiments, the PNA sequence comprises about 5 to 10 bases. In some embodiments, the PNA sequence comprises about 15 bases. In some embodiments, the PNA sequence comprises about 10 bases.

[0048] In some embodiments, the "specific binder" is a primary antibody, the primary antibody being specific for the first target. In some embodiments, the "specific binder" is a secondary antibody, and the method further comprises contacting the sample with a primary antibody specific for the first target before contacting the sample with the first conjugate, the first conjugate being specific for the first primary antibody. In some embodiments, the first detection reagent is an anti-label antibody specific for the label of the conjugate. In some embodiments, the label is a hapten, and the anti-label antibody is an anti-hapten antibody. In some embodiments, the detection reagent comprises a PNA, gamma PNA, or DNA sequence complementary to the PNA or gamma PNA sequence of the first conjugate, the complementary PNA, gamma PNA, or DNA sequence being conjugated to a reporter moiety. In some embodiments, the reporter moiety is a fluorophore. In some embodiments, the reporter moiety is a chromogen. In some embodiments, the reporter moiety is an enzyme. In some embodiments, the reporter moiety is a hapten and the method further comprises contacting the sample with an anti-hapten antibody specific for the hapten of the complementary PNA, gamma PNA or DNA sequence.

[0049] Another aspect of the present disclosure is a method for detecting a target in a sample, comprising: (a) contacting the sample with a first PNA conjugate, wherein the first PNA conjugate has formula (IIB): TIFF0007727385000008.tif34170[In the formula, The "specific binding entity" is selected from the group consisting of an antibody (e.g., a primary or secondary antibody), an antibody fragment, a drug / antibody conjugate, and a nucleic acid; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms, optionally containing one or more heteroatoms selected from O, N, or S, and including cleavable groups or moieties; A "PNA" is a PNA sequence containing at least one nucleotide with a substitution at the gamma carbon position; X is a label; Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms; m is 0; z is 0; n is an integer ranging from 1 to 12. and contacting the compound having the structure: (b) contacting the sample with a reagent (or light source, depending on the cleavable group selected) to cleave the cleavable group on the "linker"; and (c) quantifying the amount of cleaved "PNA" or gamma PNA sequence. and

[0050] One of skill in the art will recognize that the above-identified steps may be repeated multiple times using different PNA conjugates (e.g., any conjugate of formula (IIB)) to provide a multiplex assay.

[0051] In some embodiments, the "specific binding entity" is an antibody.

[0052] In some embodiments, the cleavable group is selected from the group consisting of a photocleavable group, a chemically cleavable group, or an enzymatically cleavable group, hi some embodiments, the cleavable group is a disulfide bond.

[0053] In some embodiments, X is selected from the group consisting of biotin, an enzyme, a chromogen, a fluorophore, a hapten, and a mass spectrometry tag. In some embodiments, X is biotin.

[0054] In some embodiments, the amount of PNA sequence is quantified using NanoString nCounter technology, such as that described herein. In some embodiments, the amount of PNA sequence is quantified using Gyrolab technology, such as that described herein.

[0055] In some embodiments, after cleavage of the PNA, the antibody-bound tissue sections are re-stained by conventional methods such as immunohistochemistry or immunofluorescence to allow visualization of the spatial distribution of the marker encoded by the PNA tag.

[0056] In some embodiments, the method further comprises introducing a single-stranded DNA or PNA sequence complementary to the PNA sequence of the conjugate of formula (IIB). In some embodiments, the complementary single-stranded DNA or PNA sequence is conjugated to a reporter moiety. In some embodiments, the complementary single-stranded DNA or PNA sequence is conjugated to a hapten. In some embodiments, the complementary single-stranded DNA or PNA sequence is conjugated to digoxigenin.

[0057] In some embodiments, the "specific binder" is a primary antibody, and the method further comprises introducing a secondary antibody specific for the primary antibody. In some embodiments, the secondary antibody is conjugated to a reporter moiety.

[0058] In some embodiments, the PNA sequence has at least two gamma PNA bases. In some embodiments, each of the at least two gamma PNA bases is substituted with the same amino acid, miniPEG, peptide, or polymer. In some embodiments, each of the at least two gamma PNA bases is substituted with a different amino acid, miniPEG, peptide, or polymer. In some embodiments, the amino acid, miniPEG, peptide, or polymer comprises at least one reporter moiety (e.g., fluorophore, chromogen). In some embodiments, the PNA sequence has at least three gamma PNA bases. In some embodiments, the PNA sequence has at least four gamma PNA bases.

[0059] In some embodiments, the PNA sequence comprises about 5 to 20 bases. In some embodiments, the PNA sequence comprises about 5 to 15 bases. In some embodiments, the PNA sequence comprises about 5 to 10 bases. In some embodiments, the PNA sequence comprises about 15 bases. In some embodiments, the PNA sequence comprises about 10 bases.

[0060] Unlike DNA, PNAs do not contain a charge, and therefore the bond between PNA and DNA is stronger than that between DNA and DNA, thus enabling the use of relatively short PNA sequences to label antibodies while achieving binding affinity and specificity not possible with DNA-conjugates. It is also predicted that shorter PNA sequences may help minimize interference with antibody-antigen binding and tissue nonspecific binding. Thus, the PNA-antibody conjugates disclosed herein are expected to maintain antibody binding specificity, while the PNA oligomers function as unique molecular tags that can be visualized in situ on slides (IF or IHC) by hybridization with signal-generating molecules or quantified from slides by high-throughput techniques such as NanoString technology, Gryos technology, or mass spectrometry (see Figure 1A). Additionally, virtually unlimited unique sequences / tags can be easily generated and detected under nearly identical conditions, eliminating the need to optimize individual conjugates. Importantly, a cleavable linker (photocleavable or chemically cleavable) can be placed between the PNA oligomer and the antibody (see Figure 1A), allowing for easy sample collection for off-slide protein profiling (multiplexed quantification).

[0061] 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 by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0062] [Figure 1A] An overview is provided of the structure of PNA conjugates and their use in both visualizing targets within samples (qualitative) and quantitatively assessing PNA tags cleaved from bound antibodies. [Figure 1B] The chemical structures of the heterobifunctional crosslinker and PNA sequences are provided. [Figure 2A]Figure 1 shows the characterization of antibodies and the corresponding PNA conjugates based on UV-Vis absorbance before PNA conjugation. The black and red traces represent two different antibodies as examples. [Figure 2B] Figure 1 shows the characterization of antibodies and the corresponding PNA conjugates based on UV-Vis absorbance after PNA conjugation. The black and red traces represent two different antibodies as examples. [Figure 3A] IHC detection of biotinylated PNA. The figure shows tonsillar slides treated with rabbit anti-CD45 antibody followed by PNA-conjugated GAR, GAM, and GAM, respectively. Biotin was then detected by SA-HRP and DAB deposition. [Figure 3B] IHC detection of biotinylated PNA. The figure shows tonsillar slides treated with mouse anti-Ki67 antibody followed by PNA-conjugated GAR, GAM, and GAM, respectively. Biotin was then detected by SA-HRP and DAB deposition. [Figure 3C] IHC detection of biotinylated PNA. The figure shows tonsillar slides treated without primary antibody and then with PNA-conjugated GAR, GAM, and GAM, respectively. Biotin was then detected by SA-HRP and DAB deposition. [Figure 3D] 1 shows a detection strategy using biotinylated PNA according to some embodiments of the present disclosure. [Figure 4A] IHC detection of biotinylated PNA as a membrane marker is shown. Tonsil slides were treated with a "haptenized" primary antibody (i.e., an antibody labeled with a hapten), followed by treatment with the corresponding PNA-conjugate for each hapten. Biotin in the PNA conjugate was then detected by SA-HRP and DAB deposition. [Figure 4B]IHC detection of biotinylated PNA as a nuclear marker is shown. Tonsil slides were treated with a "haptenized" primary antibody, followed by treatment with the corresponding PNA-conjugate for each hapten. The biotin in the PNA conjugate was then detected by SA-HRP and DAB deposition. [Figure 5A] Chemical cleavage of the PNA tag is shown. Tonsil slides were treated with mouse anti-Ki67 antibody and further processed by SA-HRP and DAB deposition. [Figure 5B] Chemical cleavage of the PNA tag is shown. Tonsil slides were treated with mouse anti-Ki67 antibody and further treated with approximately 20 mM TCEP prior to SA-HRP incubation and DAB deposition. [Figure 5C] Chemical cleavage of the PNA tag is shown. Tonsil slides were treated with rabbit anti-CD45 antibody and further processed by SA-HRP and DAB deposition. [Figure 5D] Chemical cleavage of the PNA tag is shown. Tonsil slides were treated with rabbit anti-CD45 antibody and further treated with approximately 20 mM TCEP prior to SA-HRP incubation and DAB deposition. [Figure 6A] Fluorescence detection of biotinylated PNA. Tonsil slides were treated with mouse anti-Ki67 antibody, followed by PNA-conjugated GAM. The slides were further incubated with SA-FITC. [Figure 6B] Fluorescence detection of biotinylated PNA. Tonsil slides were treated without primary antibody and then with PNA-conjugated GAM. Slides were further incubated with SA-FITC. [Figure 6C] 1 shows a fluorescence-based detection scheme utilizing biotinylated PNA. [Figure 7A]Quantified antibody measurements per slide based on cleaved PNA-SA-FITC fluorescence intensity are shown. Specifically, the bar graph shows the fluorescence intensity of cleaved PNA-SA-FITC from tonsillar slides treated with mouse anti-CD45, PNA-conjugated GAM, followed by SA-FITC, and finally incubated with 20 mM TCEP. Control slides were treated identically to the experimental slides but without primary antibody. [Figure 7B] Quantified measurements of antibody per slide based on cleaved PNA-SA-FITC fluorescence intensity are shown. Specifically, the standard curve used to determine the concentration of cleaved PNA-SA-FITC from experimental and control slides. Fluorescence intensities of samples and controls are shown as mean ± standard deviation (N=3). [Figure 8A] This shows the detection of PNA using fluorescently labeled complementary DNA. Tonsil slides were treated with rabbit anti-Ki67 and then with PNA-conjugated GAR. The slides were then incubated with fluorescently labeled DNA sequences complementary to the PNA tag. FITC-labeled DNA sequences were used. [Figure 8B] Figure 1 shows PNA detection with fluorescently labeled complementary DNA. A tonsillar slide was not treated with primary antibody, thus serving as a negative control. The slide was then incubated with a fluorescently labeled DNA sequence complementary to the PNA tag. FITC-labeled DNA sequences were utilized. [Figure 8C] Figure 8B shows PNA detection using fluorescently labeled complementary DNA. Tonsil slides were treated with rabbit anti-Ki67 and then with PNA-conjugated GAR. The slides were then incubated with fluorescently labeled DNA sequences complementary to the PNA tag. Rhodamine-labeled DNA sequences were used. Negative controls without primary antibody but under otherwise identical conditions are shown in Figures 8B and 8D, respectively. [Figure 8D]Figure 1 shows PNA detection with fluorescently labeled complementary DNA. A tonsil slide was not treated with primary antibody, thus serving as a negative control. The slide was then incubated with a fluorescently labeled DNA sequence complementary to the PNA tag. A rhodamine-labeled DNA sequence was utilized. [Figure 8E] 8A, 8B, 8C, and 8D are provided as schematic diagrams of the detection methods utilized in generating the images of FIGS. 8A, 8B, 8C, and 8D. [Figure 9A] Chromogenic detection with complementary haptenized DNA is shown. Tonsil slides were treated with primary mouse anti-CD20, then incubated with PNA-conjugated GAM, followed by hybridization with DIG-labeled DNA sequences complementary to the PNA tag. Finally, the slides were incubated with anti-DIG:HRP antibody and DAB deposition. [Figure 9B] Chromogenic detection with complementary haptenized DNA is shown. Tonsil slides were treated with rabbit anti-Ki67, then incubated with PNA-conjugated GAM, and then hybridized with DIG-labeled DNA sequences complementary to the PNA tag. Finally, the slides were incubated with anti-DIG:HRP antibody and DAB deposition. [Figure 9C] Shown is a tonsil slide that was treated similarly to the tissue depicted in Figures 9A and 9B, but without the primary antibody, and thus served as a negative control. [Figure 9D] 9A, 9B, and 9C are schematic diagrams of the detection methods utilized in generating the images of FIGS. 9A, 9B, and 9C. [Figure 10] 1 shows a possible synthesis scheme for a photocleavable heterobifunctional crosslinker. [Figure 11] 1 shows the chemical structures of photodegradable linkers used to synthesize photocleavable PNAs. [Figure 12A] Photocleavage of PNA is shown. Slides were treated with Ki67 and then with GAR-PL-PNA. Slides were irradiated with a handheld UV lamp for 0 minutes. [Figure 12B]Photocleavage of PNA is shown. Slides were treated with Ki67 and then with GAR-PL-PNA. Slides were irradiated with a handheld UV lamp for 5 minutes. [Figure 12C] Photocleavage of PNA is shown. Slides were treated with Ki67 and then with GAR-PL-PNA. Slides were irradiated with a handheld UV lamp for 10 minutes. [Figure 13] Selective PNA photocleavage is shown. A tonsillar slide was treated with Ki67 and then with GAR-PL-PNA. The displayed area was then irradiated with UV light from a laser capture microdissection instrument. After washing the LCM slide to remove the cleaved PNA, staining was completed with SA-HRP and DAB detection. Compared to unirradiated germinal centers on the same slide, UV-irradiated germinal centers lost color. [Figure 14A] 1 shows a flow chart outlining various methods for detecting targets in a sample using PNA conjugates of the present disclosure. [Figure 14B] 1 shows a flow chart outlining various methods for detecting targets in a sample using PNA conjugates of the present disclosure. [Figure 14C] 1 shows a flow chart outlining various methods for detecting targets in a sample using PNA conjugates of the present disclosure. [Figure 15] Stability of PNA-antibody conjugates is shown. Tonsil slides were incubated with primary antibodies (anti-CD45 and anti-Ki67), followed by GAM-PNA and GAR-PNA, respectively, and detected by SA-HRP and DAB deposition. The same antibody-PNA conjugates shown were later used to assess conjugate stability. [Figure 16] The difference between PNAs with a substituent (denoted by an "R" group, but not limited to) at the gamma carbon and PNAs without a substituent at the gamma carbon is shown. [Figure 17]Branched PNA sequences are shown. The primary PNA sequence (shown in red) has four substituted PNA bases. Substituents of the primary PNA sequence are gamma PNA sequences (shown in black). Each PNA sequence has four substituents. These substituents, shown to contain four gamma PNA nucleotides, are each substituted with a miniPEG or other reporter moiety. Each miniPEG may contain one or more labels or reporter moieties. [Figure 18] Schematic diagram showing the conjugation of an antibody to a PNA oligomer using "click chemistry" for coupling. Here, a reduced antibody is functionalized with a DBCO group and then linked to a PNA oligomer containing an azide group. The PNA oligomer may contain one or more nucleotides with a substitution at the gamma carbon. [Figure 19] Tonsil tissue was incubated with anti-Ki67 primary antibody (rabbit mAb) and GAR-PNA (click chemistry conjugated) secondary antibody and detected with SA-HRP. The image shows that the PNA was successfully conjugated to the antibody via click chemistry. [Figure 20] FIG. 1 is a schematic diagram showing the conjugation of an antibody to a PNA oligomer via a maleimide moiety. [Figure 21A] Tonsil tissue was incubated with anti-Ki67 followed by GAR-short PNA (sequence shown on previous slide) and detected with SA-HRP. Detection of biotin on the short PNA sequence. The image shows that the short PNA was successfully conjugated onto the Ab. [Figure 21B] Tonsil tissue was incubated with anti-Ki67 followed by GAR-HRP. The image shows that the short PNA was successfully conjugated onto the Ab. This is used to compare baseline detection (FIG. 21B) with GAR-short PNA-based detection (FIG. 21A). [Figure 22A]Tonsils incubated with CD3-short PNA and detected with SA-HRP are shown. IHC of short PNA conjugated with primary antibody. The concentration of Ab-short PNA conjugate used was 5 μg / mL (PNA sequence: biotin-o-CCATCTTCAG-Lys (SMCC) sequence (SEQ ID NO: 19)). [Figure 22B] Tonsils incubated with a second example of a primary antibody-short PNA conjugate (CD8-short PNA) are shown. [Figure 22C] Tonsils incubated with a third example of a primary antibody-short PNA conjugate (CD34-short PNA) are shown. [Figure 22D] Tonsils incubated with a fourth example of a primary antibody-short PNA conjugate (Ki67-short PNA) are shown. [Figure 23] Chromogenic staining with CD3 conjugated to a (10-base) PNA sequence designated sPNA2, with the sequence: biotin-o-TTAGTCCAAC-Lys(SMCC) (SEQ ID NO: 20), is shown. Tonsil tissue was stained with a PNA-conjugated Ab followed by SA-HRP to target the biotin moiety on the PNA sequence. Staining was localized, demonstrating that PNA conjugation did not alter Ab functionality. [Figure 24] Chromogenic staining with CD8 conjugated to sPNA2 (10 bases), whose PNA sequence was biotin-o-CCATCTTCAG-Lys(C6SH) (SEQ ID NO: 21), is shown. Tonsil tissue was stained with a PNA-conjugated Ab followed by SA-HRP to target the biotin moiety on the PNA sequence. Staining was localized, demonstrating that PNA conjugation did not alter Ab functionality. [Figure 25] Tonsil tissue was incubated with anti-Ki67 and GAR-short PNA, followed by DNA-DIG (a DNA sequence complementary to the short PNA tag called AB14, with DIG at its end). Anti-DIG-HRP antibody was added for detection. In this case, DNA complementary to the PNA tag was detected. Two different concentrations of complementary DNA-DIG are shown. [Figure 26] This figure shows the formation of fluorescent duplexes by DNA conjugation. The figure also shows detection by hybridization, but this time by fluorescence. The complementary DNA contains one or more fluorescent tags. Two primary antibodies, CD3 and CD8, are conjugated to two different short PNA tags, respectively. Each of the two complementary DNA sequences carries a fluorophore. [Figure 27A] This figure shows a tonsillar tissue slide incubated with a mixture of anti-CD3-short PNA1 and anti-CD8-short PNA2 (anti-CD3 and anti-CD8 conjugated to short PNA1 and short PNA2, respectively), followed by incubation with two DNA sequences complementary to sPNA1 and sPNA2, designated AB15 and AB19, respectively. AB15 carries AlexaFluor 488 at its end, while AB19 carries AlexaFluor 647 at its end. The two fluorescently labeled DNA sequences bind to their complementary PNA sequences on the tissue, resulting in a fluorescently stained tissue slide. The fluorescently stained tissue is imaged under a fluorescent microscope. The green channel (Figure 27A) shows AlexaFluor 488 on CD3-positive cells. [Figure 27B] This figure shows a tonsillar tissue slide incubated with a mixture of anti-CD3-short PNA1 and anti-CD8-short PNA2 (anti-CD3 and anti-CD8 conjugated to short PNA1 and short PNA2, respectively), followed by incubation with two DNA sequences complementary to sPNA1 and sPNA2, designated AB15 and AB19, respectively. AB15 carries AlexaFluor 488 at its end, while AB19 carries AlexaFluor 647 at its end. The two fluorescently labeled DNA sequences bind to their complementary PNA sequences on the tissue, resulting in a fluorescently stained tissue slide. The fluorescently stained tissue is imaged under a fluorescent microscope. The red channel (Figure 27B) shows AlexaFluor 647 on CD8-positive cells in the same field of view. [Figure 27C]This figure shows a tonsillar tissue slide incubated with a mixture of anti-CD3-short PNA1 and anti-CD8-short PNA2 (anti-CD3 and anti-CD8 conjugated to short PNA1 and short PNA2, respectively), followed by two DNA sequences complementary to sPNA1 and sPNA2, designated AB15 and AB19, respectively. AB15 carries AlexaFluor 488 at its end, while AB19 carries AlexaFluor 647 at its end. The two fluorescently labeled DNA sequences bind to their complementary PNA sequences on the tissue, resulting in a fluorescently stained tissue slide. The fluorescently stained tissue is imaged under a fluorescent microscope. A merge of the two images (Figure 27C) shows that all red cells (CD8 positive) are also green (CD3 positive), because all CD8-expressing cells also express CD3. Some green cells are not red because not all CD3 cells are CD8. CD8 is a subpopulation of CD3. [Figure 28] Tonsil tissue was incubated with anti-Ki67 (rabbit mAb), followed by GAR-gamma-PNA (goat anti-rabbit conjugated to gamma-PNA), and finally streptavidin-HRP, which binds to biotin at the end of the gamma-PNA. The signal was as expected, and no nonspecific signal was observed. [Figure 29A] Tonsil tissues were incubated with primary antibodies conjugated to gamma-PNA and detected with SA-HRP. Different concentrations of CD3-gamma-PNA (10 μg / mL, 5 μg / mL, and 1 μg / mL) are shown at two magnifications (20× and 40×). This demonstrates that primary antibodies can also be conjugated to gamma-PNA and detected directly. [Figure 29B] Another example of a primary antibody (CD8) conjugated to gamma PNA at different concentrations (10 μg / mL, 5 μg / mL, 1 μg / mL) is provided. [Figure 29C] Another example of a primary antibody (PD-L1) conjugated to gamma-PNA at different concentrations (1.5 μg / mL, 1 μg / mL, 0.5 μg / mL) is provided. [Figure 30] The UV absorbance of GAR-PNA and GAR-gamma PNA conjugates is shown. Here, antibodies primarily absorb light at 280 nm, whereas PNA primarily absorbs light at 260 nm. When PNA is conjugated to an antibody, the absorbance at 260 nm increases. The 260 nm / 280 nm ratio is used to evaluate conjugation efficiency. An increase in the 260 nm / 280 nm ratio indicates higher PNA loading, and gamma-PNA is expected to be more hydrophilic than PNA even without any gamma substituents. The 260 nm / 280 nm ratio of unconjugated antibody (pure antibody) is approximately 0.58, which increases to 1.1 when conjugated to PNA and to 1.3 when conjugated to gamma-PNA. [Figure 31] The UV absorbance of GAR, CD3, and CD8-short PNA conjugates is shown as a 260 nm / 280 nm ratio, which shows an increase as the PNA is conjugated to the antibody. [Figure 32] The principle of using Gryos technology for quantification of PNAs is demonstrated. [Figure 33] A schematic diagram is provided showing the steps of quantifying cleaved PNA tags using Gryos technology and re-staining the slides by IHC to allow visualization of the same markers encoded by the PNA tags. [Figure 34A] IHC staining of protein targets after cleavage of the PNA or gamma PNA sequences from the PNA-antibody conjugates is shown. [Figure 34B] IHC staining of protein targets after cleavage of the PNA or gamma PNA sequences from the PNA-antibody conjugates is shown. [Figure 35A] 1 provides a comparison of the detection of different PNA and DNA oligomers using Gryos technology. [Figure 35B] The signal-to-background (S / B) ratios of PNAs detected using Gryos technology are shown. [Figure 36A]Tonsil stained with Ki67 (rabbit Ab) followed by goat anti-rabbit (GAR) conjugated to a PNA sequence (CCATCTTCAG) is shown. The absence of signal (as shown here) indicated that the DNA did not hybridize under the experimental conditions (200 nM DNA, 37°C, 30 min). DNA PNA affinity is not strong enough to ensure hybridization with such a short PNA sequence under the experimental conditions. [Figure 36B] Shown is a tonsil stained with Ki67 (rabbit Ab) followed by goat anti-rabbit (GAR) conjugated to a gamma PNA sequence. The DNA gamma PNA affinity is believed to be strong enough to ensure hybridization, thus producing a brown signal. DETAILED DESCRIPTION OF THE INVENTION

[0063] In general, the present disclosure is directed to a conjugate, for example, a PNA conjugate, and a method of using the conjugate to detect one or more targets in a biological sample, for example, a tissue sample. Without wishing to be bound by any particular theory, it is believed that when used in an assay, the conjugate allows for the simultaneous qualitative and quantitative evaluation of multiple targets, including protein targets (see Figure 1A).

[0064] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise.

[0065] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including at least one element, but including more than one element or list of elements, and optionally, additional items not included in the list. Only terms clearly indicated otherwise, such as "only one" or "exactly one," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a plurality of elements or list of elements. In general, the term "or," as used herein, shall be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") only when preceded by an exclusive term, such as "either," "one," "only one," or "exactly one." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0066] Terms such as "comprising," "including," and "having" are used interchangeably and have the same meaning. Similarly, terms such as "comprises," "includes," and "has" are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprising" and, therefore, is interpreted as an open term meaning "at least" and not excluding additional features, limitations, embodiments, etc. Thus, for example, "an apparatus having components a, b, and c" means that the apparatus includes at least components a, b, and c. Similarly, the phrase "a method including steps a, b, and c" means that the method includes at least steps a, b, and c. Furthermore, although steps and processes may be outlined in a particular order herein, one of ordinary skill in the art will recognize that the order of steps and processes may vary.

[0067] As used in this specification and claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements of the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements and excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or not to the specifically identified elements, may optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer, in one embodiment, to at least one A, which may include more than one A, where no B is present (which may include elements other than B); in another embodiment, to at least one B, which may include more than one B, where no A is present (which may include elements other than A); in yet another embodiment, to at least one A, which may include more than one A, and at least one B, which may include more than one B (which may include other elements); etc.

[0068] As used herein, the term "alkyl" refers to a straight or branched hydrocarbon chain containing a hydrocarbon group that is fully saturated (no double or triple bonds). An alkyl group can be substituted or unsubstituted.

[0069] As used herein, the term "antibody" refers to immunoglobulins or immunoglobulin-like molecules, including, by way of example and not limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during the immune response of any vertebrate (e.g., mammals such as humans, goats, rabbits, and mice), as well as antibody fragments (such as F(ab')2 fragments, Fab' fragments, Fab'-SH fragments, and Fab fragments, as known in the art), recombinant antibody fragments (e.g., sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'2 fragments), which specifically bind to a molecule of interest (or a closely related group of molecules of interest) to the substantial exclusion of binding to other molecules. The term antibody refers to Fv proteins, such as single-chain Fv proteins ("scFv"), disulfide-stabilized Fv proteins ("dsFv"), diabodies, and triabodies (known in the art), and camelid antibodies. Furthermore, antibody refers to a polypeptide ligand comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. An antibody can be composed of a heavy chain and a light chain, each of which has a variable region called a variable heavy (VH) region and a variable light (VL) region. The VH and VL regions together are responsible for binding to the antigen recognized by the antibody. The term antibody also includes intact immunoglobulins, as well as variants and portions thereof known in the art.

[0070] As used herein, the term "antigen" refers to a compound, composition, or substance capable of specific binding 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.

[0071] As used herein, the term "biological sample" or "tissue sample" refers to any solid or liquid sample obtained from, excreted by, or secreted by any living organism, including, but not limited to, unicellular organisms such as bacteria, yeast, protozoa, and amoebas, multicellular organisms (plants or animals, including samples derived from healthy or apparently healthy human subjects or human patients suffering from a condition or disease to be diagnosed or tested, such as cancer), among others. For example, a biological sample can be, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous humor, or vitreous humor, or any bodily secretion, exudate, or transudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation), or bodily fluid obtained from a joint (e.g., a normal joint or a diseased joint). A biological sample can also be a sample obtained from any organ or tissue (including biopsy or autopsy specimens, such as tumor biopsies), or can include cells (whether primary or cultured) or media conditioned by any cell, tissue, or organ. The sample may be a tumor sample, including those from melanoma, renal cell carcinoma, and non-small cell lung cancer. In some embodiments, the sample is analyzed for a disease state, such as cancer, by detecting targets, including biomarkers (e.g., proteins or nucleic acid sequences), within the tissue sample. Described embodiments of the disclosed methods can also be adapted for samples free of abnormalities, diseases, disorders, etc., referred to as "normal" or "control" samples. For example, it may be useful to test a subject for cancer by collecting tissue samples from multiple locations; these samples can be used as controls and compared with subsequent samples to determine whether a particular cancer has spread beyond its primary origin. In some embodiments, the sample may be protein extracted and purified from a cell lysate, tissue, or whole organism by conventional protein extraction methods. In the context of the present disclosure, the extracted protein can then be mixed with a PNA-conjugated antibody, which allows the antibody to bind to a specific protein. The PNA is then released and counted to quantify the protein.

[0072] As used herein, "C" is a set of integers where "a" and "b" are integers. a From C b " refers to the number of carbon atoms in an alkyl, alkenyl, or alkynyl group, or the number of carbon atoms in a ring of a cycloalkyl, cycloalkenyl, cycloalkynyl, or aryl group, or the total number of carbon atoms and heteroatoms in a heteroalkyl, heterocyclyl, heteroaryl, or heteroalicyclyl group. That is, an alkyl, alkenyl, alkynyl, cycloalkyl ring, cycloalkenyl ring, cycloalkynyl ring, aryl ring, heteroaryl ring, or heteroalicyclyl ring can contain "a" through "b" carbon atoms, inclusively. Thus, for example, a "C1 to C4 alkyl" group refers to all alkyl groups having from 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-. When "a" and "b" are not specified for an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, or heteroalicyclyl group, the broadest range described in those definitions is to be assumed.

[0073] As used herein, a "conjugate" refers to two or more molecules (and / or materials such as nanoparticles) that are covalently linked into a larger construct.

[0074] As used herein, the term "couple" or "coupling" refers to the joining, bonding (e.g., covalent bonding), or linking of one molecule or atom to another molecule or atom.

[0075] As used herein, the term "detection probe" includes a nucleic acid probe or primary antibody that binds to a specific target (e.g., a nucleic acid sequence, a protein, etc.). Detection probes may contain labels for direct detection, such as radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent agents, haptens (including, but not limited to, DNP), and enzymes. Alternatively, detection probes may not contain a label or tag and may be detected indirectly (e.g., using a secondary antibody specific for the detection probe).

[0076] As used herein, the terms "gamma PNA" and "gamma PNA sequence" refer to any PNA sequence that includes at least one nucleotide that includes a substituent at the gamma position, i.e., at the gamma carbon position. Figure 16 illustrates the difference between PNA nucleotides and those with a substituent at the gamma carbon position, where the "R" groups are for illustrative purposes only and are non-limiting.

[0077] As used herein, the term "hapten" refers to a small molecule that can specifically bind to an antibody, but is typically substantially incapable of being immunogenic except in combination with a carrier molecule. In some embodiments, haptens include, but are not limited to, pyrazoles (e.g., nitropyrazoles); nitrophenyl compounds; benzofurazans; triterpenes; ureas (e.g., phenylureas); thioureas (e.g., phenylthioureas); rotenone and rotenone derivatives; oxazoles (e.g., oxazole sulfonamides); thiazoles (e.g., thiazole sulfonamides); coumarins and coumarin derivatives; and cyclolignans. Additional non-limiting examples of haptens include thiazoles; nitroaryls; benzofurans; triperpenes; and cyclolignans. Specific examples of haptens include dinitrophenyl, biotin, digoxigenin, and fluorescein, as well as any derivatives or analogs thereof. Other haptens are described in U.S. Patent Nos. 8,846,320; 8,618,265; 7,695,929; 8,481,270; and 9,017,954, the disclosures of which are incorporated by reference in their entireties. The haptens themselves may be suitable for direct detection, i.e., they may emit a signal suitable for detection.

[0078] As used herein, the term "halogen atom" or "halogen" means any one of the radiostable atoms in column 7 of the periodic table of the elements, for example, fluorine, chlorine, bromine, and iodine.

[0079] As used herein, the term "immunohistochemistry" refers to a method for determining the presence or distribution of an antigen in a sample by detecting the interaction of the antigen with a specific binding agent, such as an antibody. The sample is contacted with the antibody under conditions that allow antibody-antigen binding. Antibody-antigen binding can be detected by a detectable label conjugated to the antibody (direct detection) or by a detectable label conjugated to a secondary antibody that specifically binds to the primary antibody (indirect detection).

[0080] As used herein, the terms "multiplex," "multiplexing," 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 distinct nucleic acids (e.g., DNA, RNA, mRNA, miRNA) and polypeptides (e.g., proteins) individually and in any and all combinations.

[0081] As used herein, the terms "oligonucleotide," "polynucleotide," and "nucleic acid" are used herein to encompass all forms of nucleic acid molecules. This category includes, but is not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), and derivatives thereof, each with or without modification.

[0082] 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 generally the first antibody used in immunohistochemistry procedures. Thus, a primary antibody can act as a "detection probe" for detecting a target in a tissue sample.

[0083] As used herein, the term "peptide nucleic acid" or "PNA" refers to an oligonucleotide analogue in which the sugar-phosphate backbone is replaced with a pseudopeptide backbone. PNAs bind to DNA and RNA with high specificity and selectivity, resulting in PNA-RNA and PNA-DNA hybrids that are believed to be more stable than the corresponding nucleic acid complexes. The binding affinity and selectivity of PNAs for nucleic acids can be modified by introducing stereocenters (such as D-Lys-based units) into the PNA backbone. PNAs can be oligomers, linked polymers, or chimeric oligomers. Methods for the chemical synthesis and assembly of PNAs are described in U.S. Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, and 5,786,571, the disclosures of which are incorporated herein by reference in their entireties. The term "PNA" as used throughout includes PNA sequences having one or more bases substituted at gamma positions ("gamma PNAs"), i.e., the term PNA or PNA sequence includes gamma PNA or gamma PNA sequences.

[0084] As used herein, and as used throughout, the terms "reactive group" and "reactive group(s)" refer to any of a variety of groups (e.g., functional groups) suitable for linking a first unit to a second unit, as described herein. For example, the reactive group can be an amine-reactive group such as isothiocyanates, isocyanates, acyl azides, NHS esters, acid chlorides such as sulfonyl chlorides, aldehydes and glycols, epoxides and oxiranes, carbonates, arylating agents, imidoesters, carbodiimides, anhydrides, and combinations thereof. Suitable thiol-reactive functional groups include haloacetyl and alkyl halides, maleimides, aziridines, acryloyl derivatives, arylating agents, thiol-disulfide exchange reagents such as pyridyl disulfides, TNB-thiol, and disulfide reducing agents, and combinations thereof. Suitable carboxylate-reactive functional groups include diazoalkanes, diazoacetyl compounds, carbonyldiimidazole compounds, and carbodiimides. Suitable hydroxyl-reactive functional groups include epoxides and oxiranes, carbonyldiimidazole, N,N'-disuccinimidyl carbonate or N-hydroxysuccinimidyl chloroformate, periodate-oxidized compounds, enzymatic oxidation, alkyl halogens, and isocyanates. Aldehyde and ketone-reactive functional groups include hydrazines, Schiff bases, reductive amination products, Mannich condensation products, and combinations thereof. Active hydrogen-reactive compounds include diazonium derivatives, Mannich condensation products, iodination reaction products, and combinations thereof. Photoreactive chemical functional groups include aryl azides, halogenated aryl azides, benzophonones, diazo compounds, diazirine derivatives, and combinations thereof.

[0085] As used herein, the phrase "reporter moiety" refers to a molecule or material capable of producing a detectable (visual, electronic or otherwise) signal that indicates the presence (i.e., qualitative analysis) and / or concentration (i.e., quantitative analysis) of a conjugate, including a PNA conjugate, in a sample. The detectable signal can be generated by any known or yet to be discovered mechanism, including absorption, emission and / or scattering of photons (including photons of radio, microwave, infrared, visible and ultraviolet frequencies).

[0086] As used herein, the term "secondary antibody" refers to an antibody that specifically binds to a detection probe or a portion thereof (e.g., a hapten or a primary antibody), thereby forming a bridge between the detection probe and a subsequent reagent, if present (e.g., a label, an enzyme, etc.). A secondary antibody can be used, for example, to indirectly detect a detection probe, such as a primary antibody. Examples of secondary antibodies include anti-tag antibodies, anti-species antibodies, and anti-label antibodies, each of which are described herein.

[0087] As used herein, the term "specific binder" refers to a member of a specific binding pair. A specific binding pair is a pair of molecules characterized by their binding to one another to the substantial exclusion of binding to other molecules (e.g., a specific binding pair has a binding constant at least 10 times higher than the binding constant for either of the two members of the binding pair with other molecules in a biological sample). 3 M -1 Large, 10 4 M -1 Big or 10 5 M -1 (They can have large binding constants.) Examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, avidins such as streptavidin, and protein A). Specific binding moieties can also include molecules (or portions thereof) that are specifically bound by such specific binding proteins. Specific binders include the primary antibodies described above, or nucleic acid probes.

[0088] As used herein, the terms "stain," "staining," and the like generally refer to any treatment of a biological specimen to detect and / or identify the presence, location, and / or amount (e.g., concentration) of a specific molecule (e.g., lipid, protein, or nucleic acid) or a specific structure (e.g., normal or malignant cells, cytosol, nucleus, Golgi apparatus, or cytoskeleton) in the biological specimen. For example, staining can provide contrast between a specific molecule or cellular structure and the surrounding portion of the biological specimen, and the intensity of the staining can provide a measure of the amount of a specific molecule within the specimen. Staining can be used to aid in the observation of molecules, cellular structures, and organisms using not only brightfield microscopes but also other observation tools such as phase-contrast microscopes, electron microscopes, and fluorescence microscopes. Some staining performed by System 2 can be used to visualize the outlines of cells. Other staining performed by System 2 can rely on specific cellular components (e.g., molecules or structures) to be stained without or relatively little staining of other cellular components. Examples of types of staining methods performed by System 2 include, but are not limited to, histochemical methods, immunohistochemical methods, and other methods based on reactions between molecules (including non-covalent interactions), such as hybridization reactions between nucleic acid molecules. Specific staining methods include, but are not limited to, primary staining methods (e.g., H&E staining, Pap staining, etc.), enzyme-linked immunohistochemical methods, and in situ RNA and DNA hybridization methods, such as fluorescent in situ hybridization (FISH).

[0089] Whenever a group or moiety is described as "substituted" or "optionally substituted" (or "may have" or "may contain"), the group may be unsubstituted or may be substituted with one or more of the indicated substituents. Similarly, when a group is described as being "substituted or unsubstituted" if substituted, the substituents may be selected from one or more of the indicated substituents. When no substituents are indicated, the indicated "optionally substituted" or "substituted" group may each independently be alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, cyanate, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, It means that the moiety may be substituted with one or more groups selected from the group consisting of aryl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanate, thiocyanate, isothiocyanate, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, amino, ether, amino (e.g., mono- or di-substituted amino groups), and protected derivatives thereof. Any of the above groups may contain one or more heteroatoms, including O, N, or S. For example, when a moiety is substituted with an alkyl group, the alkyl group may contain a heteroatom selected from O, N, or S (e.g., —(CH—CH—O—CH—CH)—).

[0090] As used herein, the term "substantially" refers to a qualitative term indicating a degree or extent of a desired characteristic or property, either entirely or nearly entirely. In some embodiments, "substantially" means within about 20%. In some embodiments, "substantially" means within about 15%. In some embodiments, "substantially" means within about 10%. In some embodiments, "substantially" means within about 5%.

[0091] As used herein, the term "target" means any molecule whose presence, location, and / or concentration is being or can be determined. Examples of targets include nucleic acid sequences and proteins, such as those disclosed herein.

[0092] Oligomers and Conjugates One aspect of the present disclosure is a conjugate of a specific binder and an oligomer, i.e., [specific binder]-[oligomer] n (n is an integer ranging from 1 to 12). In some embodiments, the conjugate is a PNA conjugate, i.e., a conjugate of a specific binder and an oligomer comprising a PNA sequence (such as one having one or more substituents at the gamma carbon position of the PNA base). In some embodiments, the specific binder and oligomer are linked via a linker (not shown in the formula above), e.g., a linker comprising a cleavable group or moiety. In some embodiments, the oligomer comprises a gamma PNA sequence, an uncharged DNA sequence, or a DNA sequence comprising charged and uncharged bases. In other embodiments, the oligomer comprises a gamma PNA sequence. In yet other embodiments, the specific binder is an antibody and the oligomer comprises a gamma PNA sequence.

[0093] In general, the conjugates disclosed herein are suitable for use in in situ hybridization assays, including immunohistochemical assays or multiplex assays, and can be used as detection probes to detect targets in tissue samples. In some embodiments, the conjugates can hybridize to complementary PNA, DNA, or RNA or similar sequences (i) that can be directly detected; (ii) that possess a specific entity that can be directly detected, such as a fluorophore, enzyme (HRP), or (iii) that can be indirectly detected, such as by having a hapten that can be recognized by another antibody. The conjugates disclosed herein can also function as molecular "barcodes" that can be used in quantitative analysis.

[0094] In some embodiments, the conjugates of the present disclosure have the general structure of formulas (IA) and (IB): TIFF0007727385000009.tif104170[In the formula, The "specific binding entity" is selected from the group consisting of an antibody, an antibody fragment, a drug / antibody conjugate, and a nucleic acid; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; Z is selected from the group consisting of a PNA sequence comprising at least one nucleotide with a substitution at a gamma position, an uncharged DNA sequence, and a DNA sequence comprising charged and uncharged bases; X is a label (such as those described herein); Y is a spacer; m is 0 or an integer ranging from 1 to 6; z is 0 or 1; n is an integer ranging from 1 to 12].

[0095] In some embodiments, Z comprises a DNA sequence having only uncharged bases. In other embodiments, Z comprises a DNA sequence having both charged and uncharged bases. In still other embodiments, Z comprises a DNA sequence in which at least 50% of the bases in the DNA sequence are uncharged. In still other embodiments, Z comprises a PNA sequence.

[0096] In some embodiments, Z comprises a gamma PNA sequence having at least two gamma PNA bases. In some embodiments, Z comprises a gamma PNA sequence having at least three gamma PNA bases. In some embodiments, Z comprises a gamma PNA sequence having at least four gamma PNA bases.

[0097] In some embodiments, the PNA sequence comprises about 5 to 60 bases. In some embodiments, the PNA sequence comprises about 5 to 30 bases. In some embodiments, the PNA sequence comprises about 5 to 20 bases. In some embodiments, the PNA sequence comprises about 5 to 15 bases. In some embodiments, the PNA sequence comprises about 5 to 10 bases. In some embodiments, the PNA sequence comprises about 15 bases. In some embodiments, the PNA sequence comprises about 10 bases.

[0098] In some embodiments, conjugate formulas (IA) and (IB) include a linker, e.g., a multifunctional linker, designed to link a specific binding moiety to a PNA- or DNA-containing oligomer. In some embodiments, the multifunctional linker is a heterobifunctional linker, i.e., one that includes at least two different reactive functional groups (see, e.g., groups A and B defined herein). For example, the heterobifunctional linker may include a carboxylic acid group and an amine group, where one of the carboxylic acid group or the amine group is capable of forming a bond with one of the specific binding entities or PNA or DNA sequences, and the other of the carboxylic acid group or the amine group is capable of forming a bond with another of the specific binding entities or PNA or DNA sequences. In some embodiments, the "linker" includes one or more cleavable groups. In some embodiments, the one or more cleavable groups include a photocleavable moiety.

[0099] In some embodiments, a "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 40 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In other embodiments, a "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 20 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S.

[0100] In some embodiments, Y is a branched or unbranched, straight-chained or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms. In other embodiments, Y is a branched or unbranched, straight-chained or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 6 carbon atoms and optionally having one or more O, N, or S heteroatoms. In some embodiments, Y comprises a cleavable group such as those described herein with respect to a "linker." In some embodiments, n ranges from 1 to 9. In other embodiments, n ranges from 1 to 6. In some embodiments, X is a chromogenic moiety.

[0101] In some embodiments, the conjugate is a PNA conjugate having the general structure of formula (IIA): TIFF0007727385000010.tif20170[In the formula, The "specific binding entity" is selected from the group consisting of an antibody, an antibody fragment, a drug / antibody conjugate, and a nucleic acid; A "PNA oligomer" comprises a PNA sequence containing at least one nucleotide having a substitution at the gamma carbon position; n is an integer ranging from 1 to 12].

[0102] In some embodiments, a single PNA oligomer is linked to a specific binder, e.g., an antibody, nucleic acid, etc. In other embodiments, multiple PNA oligomers are linked to specific binders. In some embodiments, n is an integer ranging from 1 to 10. In other embodiments, n is an integer ranging from 1 to 8. In still other embodiments, n is an integer ranging from 1 to 6. In still other embodiments, n is an integer ranging from 2 to 6. In still other embodiments, n is an integer ranging from 1 to 4. In still other embodiments, n is an integer ranging from 2 to 4. In still other embodiments, n is at least 2. In further embodiments, n is at least 3.

[0103] A "PNA oligomer" may also include a linker or spacer designed to facilitate linkage of the PNA sequence or gamma PNA sequence to a specific binder, as further described herein. In other embodiments, a PNA oligomer may include a group that increases the water solubility of the conjugate, e.g., a linker or spacer that includes a functionality that increases the water solubility of the PNA conjugate.

[0104] In some embodiments, the "PNA oligomer" comprises at least one label that allows for direct or indirect detection of the conjugate or target.

[0105] In some embodiments, the PNA conjugate has the structure of formula (IIB): TIFF0007727385000011.tif33170[In the formula, The "specific binding entity" is selected from the group consisting of an antibody, an antibody fragment, a drug / antibody conjugate, and a nucleic acid; The linker is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; A "PNA" is a PNA sequence containing at least one nucleotide with a substitution at the gamma carbon position; X is a label; Y is a spacer; m is 0 or an integer ranging from 1 to 6; z is 0 or 1; n is an integer ranging from 1 to 12].

[0106] In some embodiments, Y is a branched or unbranched, straight-chained or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms. In some embodiments, Y is a branched or unbranched, straight-chained or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 6 carbon atoms and optionally having one or more O, N, or S heteroatoms. In some embodiments, Y comprises a cleavable group, such as those described herein for "linker."

[0107] In some embodiments, a single PNA oligomer, i.e., -(linker-PNA-[Y] Z -[X] m ) n ) is linked to a specific binder. In other embodiments, multiple PNA oligomers are linked to a specific binder. In some embodiments, n is an integer ranging from 1 to 10. In other embodiments, n is an integer ranging from 1 to 8. In still other embodiments, n is an integer ranging from 1 to 6. In still other embodiments, n is an integer ranging from 2 to 6. In still other embodiments, n is an integer ranging from 1 to 4. In still other embodiments, n is an integer ranging from 2 to 4. In still other embodiments, n is at least 2. In further embodiments, n is at least 3.

[0108] In some embodiments, the "specific binder" is a primary antibody and the PNA sequence has at least one gamma PNA base, i.e., at least one PNA base substituted at a gamma position. In some embodiments, the "specific binder" is a primary antibody and the PNA sequence has at least two gamma PNA bases. In some embodiments, the "specific binder" is a primary antibody and the PNA sequence has at least three gamma PNA bases. In some embodiments, the "specific binder" is a primary antibody and the PNA sequence has at least four gamma PNA bases.

[0109] In some embodiments, the "specific binder" is a primary antibody and the PNA sequence comprises about 10 bases. In other embodiments, the "specific binder" is a secondary antibody and the PNA sequence comprises about 15 bases. In yet other embodiments, the "specific binder" is a primary antibody and the PNA sequence comprises about 15 bases, and at least one of the nucleotides in the PNA sequence comprises a substituent at the gamma carbon position. In a further embodiment, the "specific binder" is a primary antibody and the PNA sequence comprises about 15 bases, and at least two of the nucleotides in the PNA sequence comprise a substituent at the gamma carbon position.

[0110] In some embodiments, the PNA sequence has at least two gamma PNA bases, i.e., at least two bases with a substituent at the gamma carbon position. In some embodiments, the PNA sequence has at least three gamma PNA bases. In some embodiments, the PNA sequence has at least four gamma PNA bases.

[0111] In some embodiments, the PNA sequence comprises about 5 to 60 bases. In some embodiments, the PNA sequence comprises about 5 to 30 bases. In some embodiments, the PNA sequence comprises about 5 to 20 bases. In some embodiments, the PNA sequence comprises about 5 to 15 bases. In some embodiments, the PNA sequence comprises about 5 to 10 bases. In some embodiments, the PNA sequence comprises about 15 bases. In some embodiments, the PNA sequence comprises about 10 bases.

[0112] In other embodiments, the PNA conjugate of formula (IIC) has the structure of formula (IC): TIFF0007727385000012.tif34170[In the formula, "Ab" is selected from the group consisting of a primary antibody or a secondary antibody; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; A "PNA" is a PNA sequence containing at least one nucleotide with a substitution at the gamma carbon position; X is a label; Y is a spacer; m is 0 or an integer ranging from 1 to 6; z is 0 or 1; n is an integer ranging from 1 to 12].

[0113] In some embodiments, Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 12 carbon atoms, and optionally having one or more O, N, or S heteroatoms. In some embodiments, Y comprises a cleavable group, such as those described above with respect to "linkers."

[0114] In some embodiments, one or more PNA oligomers, i.e., -(linker-PNA-[Y] z -[X] m ) n ) is linked to a primary or secondary antibody to form a PNA-antibody conjugate. In some embodiments, at least one PNA oligomer is linked to a primary antibody. In some embodiments, at least one PNA oligomer is linked to a secondary antibody. In some embodiments, n is an integer ranging from 1 to 10. In other embodiments, n is an integer ranging from 1 to 8. In still other embodiments, n is an integer ranging from 1 to 6. In still other embodiments, n is an integer ranging from 2 to 6. In still other embodiments, n is an integer ranging from 1 to 4. In still other embodiments, n is an integer ranging from 2 to 4.

[0115] The number of PNA oligomers that can be linked to any particular primary or secondary antibody will, of course, depend on the particular antibody selected and its physical and / or chemical properties. In some embodiments, the degree of labeling of oligomers per antibody ranges from about 2 to about 10. In other embodiments, the degree of labeling is greater than about 1. In other embodiments, the degree of labeling ranges from about 2 to about 6. In yet other embodiments, the degree of labeling is about 4. A relatively low degree of labeling is believed to prevent or reduce any adverse effects on antibody functionality (e.g., antigen binding or long-term stability of the labeled antibody). Again, antibody stability is believed to be largely dependent on the antibody itself. Thus, the number of PNA oligomers per antibody may depend on the antibody's ability to withstand functionalization. Indeed, it is even possible to include PNA oligomers containing multiple labels (as described further herein). For example, PNA oligomers may contain one or more fluorophores and / or haptens. In some embodiments, one or more haptens may be used for detection. In some embodiments, one or more fluorophores can be used to monitor binding of the PNA to the antibody.

[0116] In some embodiments, the PNA sequence has at least two gamma PNA bases. In some embodiments, the PNA sequence has at least three gamma PNA bases. In some embodiments, the PNA sequence has at least four gamma PNA bases.

[0117] In some embodiments, the PNA sequence comprises about 5 to 60 bases. In some embodiments, the PNA sequence comprises about 5 to 30 bases. In some embodiments, the PNA sequence comprises about 5 to 20 bases. In some embodiments, the PNA sequence comprises about 5 to 15 bases. In some embodiments, the PNA sequence comprises about 5 to 10 bases. In some embodiments, the PNA sequence comprises about 15 bases. In some embodiments, the PNA sequence comprises about 10 bases.

[0118] In some embodiments, the PNA oligomer has the structure of formula (III): TIFF0007727385000013.tif21170[In the formula, T is a group having a terminal reactive moiety; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; A "PNA" is a PNA sequence containing at least one nucleotide with a substitution at the gamma carbon position; X is a label; Y is a spacer; m is 0 or an integer ranging from 1 to 6; z is 0 or 1].

[0119] In some embodiments, the PNA sequence has at least two gamma PNA bases. In some embodiments, the PNA sequence has at least three gamma PNA bases. In some embodiments, the PNA sequence has at least four gamma PNA bases. In some embodiments, the PNA sequence has at least one gamma PNA base, and at least one gamma PNA base is directly or indirectly conjugated to a reporter moiety.

[0120] PNA oligomers can be linked to any portion of an antibody. Three functional groups in antibodies are sites for covalent modification: amines (-NH2), thiol groups (-SH), and carbohydrate residues (Shrestha D et al., 2012). Therefore, any PNA oligomer disclosed herein can be linked to an amine residue, a thiol residue, a carbohydrate residue, or any combination thereof. In some embodiments, a PNA oligomer is linked to the Fc portion of an antibody. In other embodiments, a PNA oligomer is linked to the hinge region of an antibody. In some embodiments, a PNA oligomer is linked to one or more of the Fc regions of an antibody and one or more of the hinge regions of an antibody. Indeed, any combination is contemplated by the present disclosure.

[0121] Amino groups are generally preferred, mainly because these moieties are abundant in antibodies. However, the randomness of amino groups brings the risk that antibodies may become inactive. (Adamczyk M et al., 1999, Bioconjug Chem; Jeanson A et al., 1988, J Immunol Methods; Vira S et al., 2010, Anal Biochem; Pearson JE et al., 1998, J Immunol Methods). In some embodiments, one or more PNA oligomers are linked to the amino group of an antibody.

[0122] On the other hand, under appropriate reaction conditions, sulfhydryl labeling results in highly specific targeting of the disulfide bond between the two heavy chains of an antibody in the hinge region. Because the hinge region is far from the antigen-binding site, this modification is thought to better preserve the binding affinity of the antibody. In some embodiments, one or more PNA oligomers are linked to the thiol group of an antibody.

[0123] Conjugation with the carbohydrate moiety present in the Fc portion of an antibody is similar to conjugation with a thiol group, and as a result, modification occurs at the -CHO group, which is away from the antigen-binding site. Again, without wishing to be bound by a particular theory, conjugation with carbohydrates is thought to have less adverse effects on the binding affinity of antibodies. The degree of labeling varies depending on the glycosylation state of specific antibodies. However, loss of antibody affinity was still reported by Jeanson A et al., 1988, J Immunol Methods. In some embodiments, one or more PNA oligomers are linked to the carbohydrate group of an antibody.

[0124] In some embodiments, T is a reactive group capable of forming a direct bond with a functional group of a specific binder, e.g., an amino group of an antibody, or an indirect bond via an azide on a PNA sequence attached to a thiol group of an antibody via a linker, e.g., a DBCO-maleimide bifunctional linker. In some embodiments, T is an NHS ester, a thiol, a maleimide, or an azide group.

[0125] In other embodiments, T contains reactive functional groups capable of participating in nucleophilic substitution (e.g., reaction of amines and alcohols with acyl halides, activated esters), electrophilic substitution (e.g., enamine reactions), and addition to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are discussed, for example, in March, ADVANCED ORGANIC CHEMISTRY, 3rd ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982.

[0126] In some embodiments, the reactive functional group is a carboxylic acid, an activated ester of a carboxylic acid, a carbodiimide, a sulfonyl halide, an acyl halide, a silyl halide, an acyl azide, an acyl nitrile, an acrylamide, an amine, an aldehyde, an alkyl halide (wherein the halide can be later displaced with a nucleophilic group, such as an amine, a carboxylate anion, a thiol anion, a carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom), an aryl halide, a sulfonic acid Reactive functional groups include alkyls, sulfonate esters, anhydrides, azides, aziridines, diazoalkanes, haloacetamides, halotriazines, hydrazines, hydroxylamines, isocyanates, isothiocyanates, maleimides, phosphoramidates, thiols (which, in some embodiments, can be converted to disulfides reacted with acyl halides or metal-bound), hydroxyls (which can be converted to esters, ethers, aldehydes, etc.), hydrazines, and alkynes (which can undergo, for example, cycloaddition, acylation, Michael addition). In some embodiments, the reactive functional group is a carboxyl group or various derivatives thereof, including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters. In some embodiments, the reactive functional group is a dienophile group capable of participating in a Diels-Alder reaction, such as a maleimide group. In some embodiments, the reactive functional group is an aldehyde or ketone group, such that subsequent derivatization is possible via the formation of a carbonyl derivative, such as an imine, hydrazone, semicarbazone, or oxime, or via mechanisms such as Grignard addition or alkyllithium addition.

[0127] In other embodiments, the reactive functional group is selected from carboxylic acids, activated esters of carboxylic acids, sulfonyl halides, acyl halides, amines, alkyl or aryl halides, anhydrides, azides, haloacetamides, halotriazines, hydrazines, isocyanates, isothiocyanates, maleimides, phosphoramidates, thiols, hydroxyls, and alkynes. In yet other embodiments, the reactive functional group is selected from carboxylic acids, activated esters of carboxylic acids, amines, azides, haloacetamides, hydrazines, isocyanates, maleimides, and alkynes.

[0128] PNA sequence In some embodiments, the PNA sequence is homogeneous, i.e., contains a single nucleotide type. In other embodiments, the PNA sequence is heterogeneous, i.e., contains multiple nucleotide types, and the nucleotides can be organized randomly or in repeating groups. In still other embodiments, the PNA sequence can be designed to encode specific information, e.g., a barcode, as opposed to functioning solely as a carrier.

[0129] In some embodiments, the PNA sequence comprises 2 to 60 bases. In other embodiments, the PNA sequence comprises 2 to 50 bases. In still other embodiments, the PNA sequence comprises 2 to 40 bases. In further embodiments, the PNA sequence comprises 2 to 40 bases. In still further embodiments, the PNA sequence comprises 1 to 30 bases. In further embodiments, the PNA sequence comprises 1 to 20 bases. In other embodiments, the PNA sequence comprises 20 to 40 bases. In still other embodiments, the PNA sequence comprises 20 to 30 bases. In still other embodiments, the PNA sequence comprises 30 to 40 bases. In still other embodiments, the PNA sequence comprises 5 to 20 bases. In still other embodiments, the PNA sequence comprises 5 to 15 bases. In still other embodiments, the PNA sequence comprises 8 to 12 bases. In still other embodiments, the PNA sequence comprises 12 to 18 bases. In still further embodiments, the PNA sequence comprises about 10 bases. In a further embodiment, the PNA sequence comprises about 15 bases. In a further embodiment, the PNA sequence comprises at least 10 bases. In a further embodiment, the PNA sequence comprises at least 150 bases.

[0130] In some embodiments, one or more PNA nucleotides of any PNA sequence are independently derivatized or substituted at a site on the gamma carbon of the nucleotide (see, e.g., Figure 16). mGamma PNAs (compared to PNAs without gamma substituents) are believed to be about 5°C to about 8°C higher per single nucleotide substitution, resulting in more sequence-specific binding with higher affinity. Gamma PNAs (compared to PNAs without gamma substituents) are also believed to offer several advantages, such as improved solubility, reduced self-aggregation, more stable PNA-DNA duplex formation, and flexibility for multiple labeling and other functionalization. For example, the gamma substituents can be derivatized with amino acids such as lysine, alanine, arginine, glutamic acid, and the like. In some embodiments, the one or more charged moieties are lysine or a derivative thereof. In other embodiments, the one or more charged moieties are l-(S)-lysine. In yet other embodiments, the one or more charged moieties are d-(R)-lysine. In further embodiments, the gamma substituents can be derivatized with thialysine. In some embodiments, the introduction of lysine further facilitates chemical conjugation.

[0131] In some embodiments, one or more PNA nucleotides (such as those described herein) of any PNA sequence are independently derivatized at a charged site on the gamma carbon of the PNA base. In some embodiments, one or more of the charged moieties is lysine. In some embodiments, one or more of the charged moieties is a peptide (e.g., a peptide having 2 to 20 amino acids, a peptide having 2 to 10 amino acids, a peptide having 2 to 8 amino acids, or a peptide having 1 to 5 amino acids). For example, the peptide may be lysine-guanine-lysine or lysine-guanine-guanine-guanine-lysine. As another example, the peptide may be lysine-[U] q -lysine, where U represents an amino acid and q is 0 or an integer ranging from 1 to 20. In instances where q is 1 or more, U can represent homogeneous or heterogeneous short peptide sequences, i.e., those containing the same or different amino acids. In some embodiments, U is selected from lysine, alanine, arginine, guanine, glutamic acid, or any combination thereof.

[0132] In some embodiments, one or more PNA nucleotides in any PNA sequence are independently substituted with a polymer at the gamma position. In some embodiments, the polymer at least partially confers hydrophilicity to the PNA sequence or PNA oligomer. In some embodiments, one or more PNA nucleotides are independently derivatized with a short-chain oligoethylene moiety at at least one gamma carbon of the PNA sequence.

[0133] In some embodiments, one or more PNA nucleotides of any PNA sequence are independently substituted with a "miniPEG" at the gamma carbon position. In the context of the present disclosure, the term "miniPEG" refers to a single polyethylene glycol (PEG) unit or a polymer of PEG containing 2 to 50 PEG monomers. According to one embodiment, the term miniPEG includes, but is not limited to, -CH2-(OCH2-CH2) q and -OP group, where subscript q is an integer between 1 and 50, and P is selected from the group consisting of H, (C-C) alkyl, (C-C) alkenyl, (C-C) alkynyl, (C-C) aryl, (C-C) cycloalkyl, (C-C) aryl(C-C) alkylene, and (C-C) cycloalkyl(C-C) alkylene. Examples of miniPEG units include, but are not limited to, -CH-(OCH-CH) 1~45 OH, -CH2-(OCH2-CH2) 1~40 OH, -CH2-(OCH2-CH2) 1~35 OH, -CH2-(OCH2-CH2) 1~30 OH, -CH2-(OCH2-CH2) 1~25 OH, -CH2-(OCH2-CH2) 1~20 OH, -CH2-(OCH2-CH2) 1~15 OH, -CH2-(OCH2-CH2) 1~10 OH, and -CH2-(OCH2-CH2) 1~5 An example is an OH group.

[0134] A further example of the class minPEG is -CH2-(OCH2-CH2) 1~45 O(C1-C8) alkyl, -CH2-(OCH2-CH2) 1~40 (C1-C8) alkyl, -CH2-(OCH2-CH2) 1~35 O(C1-C8) alkyl, -CH2-(OCH2-CH2) 1~30 O(C1-C8) alkyl, -CH2-(OCH2-CH2) 1~25 O(C1-C8) alkyl, -CH2-(OCH2-CH2) 1~20 O(C1-C8) alkyl, -CH2-(OCH2-CH2) 1~15 O(C1-C8) alkyl, -CH2-(OCH2-CH2) 1~10 O(C1-C8) alkyl, and -CH2-(OCH2-CH2) 1~5 It is an O(C1-C8) alkyl group.

[0135] Additional examples of gamma-substituted PNAs and methods for their synthesis are disclosed in U.S. Patent Application Publication No. 2016 / 0096867, the disclosure of which is incorporated herein by reference in its entirety.

[0136] In some embodiments, the substituent at the gamma position further comprises at least one reporter moiety directly or indirectly conjugated thereto. In some embodiments, the gamma position may be directly substituted with a reporter moiety. In other embodiments, the gamma position may be indirectly substituted with a reporter moiety, such as via a short linker group. For example, if the gamma position is substituted with a lysine residue, the reporter moiety may be conjugated to the lysine residue.

[0137] In some embodiments, any substituent at the gamma carbon position comprises more than one reporter moiety. By way of example only, if the gamma position is substituted with a miniPEG, polymer, or peptide, then multiple reporter moieties may be conjugated to the miniPEG, polymer, or peptide substituent (see, for example, Figure 17). In some embodiments, multiple PNA nucleotides each have a substituent at the gamma position, resulting in a branched PNA oligomer, with each branch comprising a miniPEG, polymer, or peptide with multiple reporter moieties. In some embodiments, the gamma carbon of the PNA oligomer may be substituted with another PNA sequence or a gamma PNA sequence.

[0138] Non-limiting examples of PNA sequences are provided below. In one embodiment, a PNA can have the sequence GTCAACCATCTTCAG (SEQ ID NO: 1). In another embodiment, a PNA can have the sequence TTAGTCCAACTGGCA (SEQ ID NO: 2). In another embodiment, a PNA can have the sequence CATTCAAATCCCCGA (SEQ ID NO: 3). In another embodiment, a PNA can have the sequence CCATCTTCAG (SEQ ID NO: 4). In another embodiment, a PNA can have the sequence TTAGTCCAAC (SEQ ID NO: 5). In another embodiment, a PNA can have the sequence CATTCAAATC (SEQ ID NO: 6). In another embodiment, a PNA can have the sequence CATCCTGCCG (SEQ ID NO: 7). Because PNA bases are not charged (like DNA bases), PNAs are considered hydrophobic compared to DNA. PNA hydrophobicity is also considered proportional to the number of bases making up the PNA sequence. Thus, the more bases a PNA sequence has, the more hydrophobic it will be. As a result, for example, a 15-base PNA sequence is more hydrophobic than a 10-base PNA sequence. Because hydrophobic PNA sequences are not easily dissolved in aqueous solution, they are also considered to be more difficult to conjugate to antibodies. Furthermore, conjugation of hydrophobic PNA sequences to antibodies can cause the conjugate to malfunction, which manifests as nonspecific binding of the conjugate to tissue. Reducing the number of bases in the PNA sequence (e.g., from 10 to 15 bases) can result in higher PNA loading and more stable conjugates.

[0139] Any of SEQ ID NOs: 1-7 may be modified by one of skill in the art so that a substituent is present at the gamma position of any of the PNA bases. Of course, each gamma position may be modified with the same or different substituents (e.g., one base may be substituted with a miniPEG having a first molecular weight, while another base may be substituted with another miniPEG having a second molecular weight, or may instead be substituted with lysine). By way of example, any of the sequences listed herein may be modified with one or more substituents at the gamma position, e.g., GT * CAA * CCA* TCT * TCA * G (where " * " indicates a PNA nucleotide containing a substituent at the gamma carbon position. As another example, SEQ ID NO: 2 may contain one or more substituents at the gamma position, e.g., TT * AGT * CCA * ACT * GGC * A (where " * " indicates a PNA nucleotide containing a substituent at the gamma carbon position. As yet another example, SEQ ID NO: 2 may contain one or more substituents at the gamma position, e.g., T * TAGTC * CA * ACT * GGC * A (where " * " indicates a PNA nucleotide containing a substituent at the gamma carbon. Of course, any of the gamma substituents may contain one or more reporter moieties.

[0140] Linker Generally, and as described above with respect to Formulas (IA), (IB), (IIA), (IIB), (IIC), and (III), a "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S. Generally, the linker has a molecular weight ranging from about 1 g / mol to about 3000 g / mol. In other embodiments, the linker has a molecular weight ranging from about 20 g / mol to about 200 g / mol. In some embodiments, the linker has a length ranging from about 0.5 nm to about 20 nm. In other embodiments, the linker has a length of less than about 15 nm. In still other embodiments, the linker has a length of less than about 10 nm.

[0141] In some embodiments, the "linker" has the structure shown in formula (IVA): TIFF0007727385000014.tif47170 [wherein d and e are each independently an integer ranging from 2 to 20; Q is a bond, O, S, or N(R c )(R d ) and R a and R b are independently H, a C1-C4 alkyl group, F, Cl, or N(R c )(R d ) and R c and R d is independently CH or H; A and B are independently branched or unbranched, straight-chain 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, d and e are integers ranging from 2 to 6. In some embodiments, at least one of A or B comprises a cleavable moiety, as described in more detail herein.

[0142] In some embodiments, the "linker" has the structure shown in formula (IVB): TIFF0007727385000015.tif39170[In the formula, d and e are each independently an integer ranging from 2 to 20; Q is a bond, O, S, or N(R c )(R d ) and; R c and R d are independently CH or H; A and B are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having from 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.

[0143] In some embodiments, the "linker" has the structure shown in formula (IVC): TIFF0007727385000016.tif40170[In the formula, d and e are each independently an integer ranging from 2 to 20; A and B are independently branched or unbranched, straight chain 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 other embodiments, d and e are integers ranging from 2 to 15. 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.

[0144] In some embodiments, the "linker" comprises a solubilizing group, such as a polyethylene glycol (PEG) group, to increase the water solubility of the PNA conjugate. In some embodiments, the linker comprises about 2 to about 24 PEG groups. In some embodiments, the linker comprises about 2 to about 18 PEG groups. In other embodiments, the linker comprises about 2 to about 12 PEG groups. In yet other embodiments, the linker comprises about 2 to about 6 PEG groups. In yet other embodiments, the linker comprises 4 PEG groups. In yet other embodiments, the linker comprises 8 PEG groups. In yet other embodiments, the linker comprises 12 PEG groups. In yet other embodiments, the linker comprises 16 PEG groups. In yet other embodiments, the linker comprises 24 PEG groups. Without wishing to be bound by any particular theory, it is believed that the introduction of such alkylene oxide linkers increases the hydrophilicity of the PNA conjugate. Those skilled in the art will recognize that as the number of alkylene oxide repeating units in the linker increases, the hydrophilicity of the PNA conjugate may also increase. Additional heterobifunctional polyalkylene glycol linkers useful in practicing certain disclosed embodiments of the present disclosure are described in U.S. Patent Application Nos. 2006 / 0246542 and 2009 / 00181398, the disclosures of each of which are incorporated herein by reference in their entireties.

[0145] In some embodiments, groups A and B comprise moieties capable of forming bonds with a group of specific binding entities or a group of PNA sequences. In some embodiments, one or both of A or B are carbonyl-reactive groups. Suitable carbonyl-reactive groups include hydrazine, hydrazine derivatives, and amines. In other embodiments, one or both of A or B are amine-reactive groups. Suitable amine-reactive groups include active esters such as NHS or sulfo-NHS, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, imide esters, anhydrides, and the like. In further embodiments, one or both of A or B are thiol-reactive groups. Suitable thiol-reactive groups include non-polymerizable Michael acceptors, haloacetyl groups (such as iodoacetyl), alkyl halides, SPDP (succinimidyl 3-(2-pyridyldithio)propionate), maleimides, aziridines, acryloyl groups, vinyl sulfones, benzoquinones, aromatic groups, which can undergo nucleophilic substitution, such as fluorobenzene groups (such as tetra- and pentafluorobenzene groups), and disulfide groups, such as pyridyl disulfide groups, and thiols activated with Ellman's reagent. Other suitable reactive groups for the "T" moiety are described above.

[0146] In some embodiments, A and / or B comprise an ultraviolet (UV) or visible light photocleavable group. For example, a group that can be cleaved upon exposure to an electromagnetic radiation source having a wavelength of about 200 nm to about 400 nm (UV) or about 400 nm to about 800 nm (visible) can be introduced. In some embodiments, the UV or visible light photocleavable group is an arylcarbonylmethyl group (including 4-acetyl-2-nitrobenzyl, dimethylphenacyl (DMP), 2-(alkoxymethyl)-5-methyl-α-chloroacetophenone, 2,5-dimethylbenzoyloxirane, and a benzoin group: 3',5'-dimethoxybenzoin (DMB)), an O-nitrobenzyl group (including 1-(2-nitrophenyl)ethyl (NPE), 1-(methoxymethyl)-2-nitrobenzene, 4,5-dimethoxy-2 o-nitrobenzyl (DMNB); α-carboxynitrobenzyl (α-CNB), o-nitro-2-phenethyloxycarbonyl groups, including 1-(2-nitrophenyl)ethyloxycarbonyl and 2-nitro-2-phenethyl derivatives, and o-nitroanilides such as acylated 5-bromo-7-nitroindoline; coumarin-4-ylmethyl groups, including 7-methoxycoumarin derivatives; and arylmethyl groups, including o-hydroxyarylmethyl groups.

[0147] In other embodiments, A and / or B comprise a near-infrared photocleavable group. In some embodiments, a group that can be cleaved when exposed to an electromagnetic radiation source having a wavelength of about 700 nm to about 1000 nm can be introduced. Suitable near-infrared photocleavable groups include cyanine groups, including heptamethine cyanine substituted with C4-dialkylamine. Without wishing to be bound by any particular theory, it is believed that the introduction of a photocleavable linker allows spatial control over PNA release and ultimately allows quantitative measurement of marker expression.

[0148] In yet other embodiments, A and / or B comprise chemically cleavable groups that can be cleaved by different chemical reactants, including reducing agents, or by pH-induced changes (e.g., cleavage of the group at a pH below 7). Suitable chemically cleavable groups include disulfide-based groups; diazobenzene groups (including the 2-(2-alkoxy-4-hydroxy-phenylazo)benzoic acid scaffold, which is sensitive to sodium dithionite); ester-based groups (high pH); and acid-sensitive linkers (such as dialkoxydiphenylsilane linkers or acylhydrazones). Vicinal diol-cleavable linkers can be cleaved with NaIO, as described, for example, in "A simple and effective cleavable linker for chemical proteomics applications," Mol Cell Proteomics, January 2013;12(1):237-44. doi:10.1074 / mcp.M112.021014. Epub 2012 October 1. In a further embodiment, A and / or B comprise an enzymatically cleavable linker. Suitable enzymatically cleavable groups include trypsin-cleavable groups and V8 protease-cleavable groups.

[0149] In some embodiments, the multifunctional linker is selected to be capable of being orthogonally protected and deprotected, allowing one of skill in the art to conjugate either the specific binding entity or the PNA moiety to the multifunctional linker at one time, thereby preventing undesired side reactions or by-products.

[0150] sign In some embodiments, X is selected from a hapten, a fluorophore, a chromogen, an enzyme, a ligand, a phosphorescent or chemiluminescent agent, a quantum dot, a mass spectrometry tag, or any other suitable entity. The type of label selected will depend on the PNA oligomer or PNA conjugate being synthesized and the ultimate role of the PNA conjugate after conjugation to an appropriate specific binder. For example, in some embodiments, the label can be selected so that the label can be directly detected when the PNA oligomer is conjugated to an antibody (e.g., fluorescein or a fluorescein derivative or analog). In other embodiments, the label can be selected so that the label can be indirectly detected when the PNA oligomer is conjugated to an antibody (e.g., detection of a hapten label by using a secondary antibody specific to the hapten, where the secondary antibody is conjugated to a detectable moiety). Guidance on the selection of suitable labels for various purposes is discussed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences (1987), the disclosures of which are incorporated herein by reference.

[0151] Fluorophores belong to several general chemical classes, including coumarins, fluoresceins (or derivatives and analogs of fluorescein), rhodamines, resorufins, luminophores, and cyanines. Additional examples of fluorescent molecules can be found in *The Handbook - A Guide to Fluorescent Probes and Labeling Technologies*, Molecular Probes, Eugene, OR.

[0152] Where the label comprises an enzyme, a detectable substrate (i.e., a substrate for the enzyme), such as a chromogenic moiety, fluorogenic compound, or luminescent compound, can be used in combination with the enzyme to generate a detectable signal (a wide variety of such compounds are commercially available, for example, from Invitrogen Corporation, Eugene, OR). Specific examples of chromogenic compounds / substrates include diaminobenzidine (DAB), 4-nitrophenyl phosphate (pNPP), Fast Red, bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), BCIP / NBT, Fast Red, AP Orange, AP Blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline sulfonic acid] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-β-D-galactopyranoside (ON), and nitrophenyl-β-D-galactopyranoside (ON). PG), o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal), p-nitrophenyl-α-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue, and tetrazolium violet.

[0153] Alternatively, enzymes can be used in metallographic detection schemes. Metallographic detection methods involve the use of enzymes such as alkaline phosphatase in combination with water-soluble metal ions and a redox-inactive substrate for the enzyme. In some embodiments, the substrate is converted by the enzyme into a redox-active agent, which reduces the metal ions, thereby forming a detectable precipitate. (See, for example, U.S. Patent Application No. 11 / 015,646, filed December 20, 2004; PCT Publication No. 2005 / 003777; and U.S. Patent Application Publication No. 2004 / 0265922, each of which is incorporated herein by reference.) Metallographic detection methods involve the use of an oxidoreductase enzyme (e.g., horseradish peroxidase) in combination with water-soluble metal ions, an oxidizing agent, and a reducing agent to also form a detectable precipitate. (See, for example, U.S. Patent No. 6,670,113, incorporated herein by reference.)

[0154] Exemplary haptens are disclosed herein. An example of the use of mass spectrometry to analyze PNA sequences is described in "Peptide nucleic acid characterization by MALDI-TOF mass spectrometry," Anal Chem. 1996 Sep. 15;68(18):3283-7.

[0155] In some embodiments, X is selected from the group consisting of dinitrophenyl, biotin, digoxigenin, fluorescein, rhodamine, or a combination thereof. In other embodiments, X is selected from the group consisting of oxazole, pyrazole, thiazole, nitroaryl, benzofuran, triterpene, urea, thiourea, rotenoid, coumarin, cyclolignan, or a combination thereof. In still other embodiments, X is selected from the group consisting of 5-nitro-3-pyrazolecarbamide, 2-(3,4-dimethoxyphenyl)quinoline-4-carboxylic acid), 3-hydroxy-2-quinoxalinecarbamide, 2,1,3-benzoxadiazole-5-carbamide, and 2-acetamido-4-methyl-5-thiazolesulfonamide. In still other embodiments, X can be selected from any of the haptens, chromophores, fluorophores, and enzymes further described herein (see, e.g., those listed herein as "reporter moieties").

[0156] Other suitable labels are described in PCT Publication No. WO / 2018 / 002015, the disclosure of which is incorporated herein by reference in its entirety. For example, suitable labels include multi-dye conjugates having at least two chromophores linked directly or indirectly to each other.

[0157] Of course, in some embodiments, the PNA conjugate does not include any label, ie, the PNA oligomer portion of the PNA conjugate terminates in a nucleotide.

[0158] Synthesis of PNA conjugates PNA conjugates can be synthesized by any means known to those skilled in the art. In some embodiments, PNA or gamma PNA oligomers (e.g., those containing a linker or reporter moiety) are linked to specific binders via heterobifunctional crosslinkers, such as crosslinkers with NHS ester groups (e.g., SPDP-PEG8-NHS), as shown in Figure 1B. Examples of heterobifunctional crosslinkers include DBCO-PEGn-maleimide, DBCO-PEGn-NHS, N3-PEGn-NHS, or N3-PEGn-maleimide (where n ranges from 0 to 20), as shown in Figure 18. Non-limiting examples of PNA sequences suitable for conjugation include the following: PNA 1: 5'-Biotin-o-GTCAACCATCTTCAG-Lysine(C6SH)-3' (SEQ ID NO: 8) PNA 2: 5'-biotin-o-TTAGTCCAACTGGCA-Lys(C6SH)-3' (SEQ ID NO: 9) PNA 3: 5'-biotin-o-CATTCAAATCCCCGA-PL-Lys(C6SH)-3' (SEQ ID NO: 10) PNA 4: 5'-biotin-o-CTGAAGATGGTTTAC-Lys(C6SH)-3' (SEQ ID NO: 11) PNA 5: 5'-Alexa488-o-CATCCTGCCGCTATG-Lys(C6SH)-3' (SEQ ID NO: 12) PNA 6: 5'-Biotin-o-GTCAACCATCTTCAG-Arg-o-Cys-3' (SEQ ID NO: 13)

[0159] Although the PNA sequences of the above identified sizes each have 15 bases, one of skill in the art will recognize that similarly functionalized PNA sequences can contain any number of bases, such as 10 bases. For example, a PNA sequence can be sPNA4:biotin-o-CCATCTTCAG-Lys(C6SH).

[0160] In some embodiments, the NHS side of the crosslinker is used to bind to amine groups on the antibody to form amide bonds; whereas the (succinimidyl 3-(2-pyridyldithio)propionate) ("SPDP") side of the crosslinker reacts with sulfhydryl groups at the 3' end (C-terminus) of the PNA sequence to form disulfide bonds. In some embodiments, the disulfide bonds can be chemically cleaved using a reducing agent.

[0161] As described herein, in some embodiments, cleaved PNA sequences can be detected and measured using the NanoString nCounter platform. For example, the PNA sequence can contain biotin at the 5' end (N-terminus) to enable direct immobilization to a streptavidin surface without the need for a capture strand (Figure 1B). This modification is expected to enable the use of much shorter PNA sequences (approximately 15 bases) that hybridize directly to the reporter strand and are further analyzed by the nCounter platform. In some embodiments, PNA sequences with approximately 15 bases provide sufficient binding affinity and specificity for the reporter sequence during detection. While not wishing to be bound by any particular theory, it is believed that the Tm of a 15-mer PNA-DNA complex is similar to that of a 50-mer DNA-DNA complex. Examples of PNA sequences suitable for ligation are provided below:

[0162] In some embodiments, a photocleavable (PC) linker can be introduced between the PNA sequence and a specific binder (e.g., an antibody) to enable light-triggered release of the PNA (see the groups identified as A or B as defined herein). In some embodiments, the PNA sequence can be synthesized with a photocleavable linker: biotin-o-CATTCAAATCCCCGA-PL-Lys(C6SH) (SEQ ID NO: 10). After photoirradiation, the PNA sequence can be released intact and can be measured using any of the techniques described herein. Alternatively, a photocleavable bifunctional linker can be synthesized (Figure 10) and used to link the PNA to an antibody.

[0163] Multiple PNA oligomers (having the same or different sequences) can be conjugated to antibody via different linkers.These PNA oligomers can have the same or different cleavable groups (for example, the PNA sequences can be the same, but the linkers that introduce the cleavable groups can be different).Some PNA oligomers can be cleavable, while others are not; or some can be cleavable under certain conditions, while others are not cleavable under the same conditions.

[0164] Introducing a photocleavable linker into the PNA during synthesis of the PNA conjugate is advantageous because it is more time- and cost-effective and ensures that the photocleavable linker is incorporated into all PNA oligomers. Alternatively, a photocleavable bifunctional linker can be synthesized (Figure 10) and used to link the PNA sequence to the antibody. This approach is advantageous because the photocleavable bifunctional linker can be used to link any PNA sequence to an antibody, even if the PNA was not designed to be photocleavable. While not wishing to be bound by any particular theory, this approach may provide some flexibility in terms of using the same PNA sequence as a regular (non-photocleavable) and photocleavable antibody tag. In some embodiments, the disulfide bond remains present in the synthesized photocleavable PNA, allowing for both photocleavage and chemical cleavage. In some embodiments, a biotin moiety is also retained, allowing for detection of SA-HRP and DAB on the slide, as described further herein.

[0165] In some embodiments, PNA sequences can be introduced to specific binding moieties and / or linkers using "click chemistry," as shown in Figure 18. Non-limiting examples of PNA sequences with reactive groups (e.g., azides) capable of undergoing a "click" reaction are provided below: PNA 7: 5'-Biotin-O-GTCAACCATCTTCAG-Lys(eg3-N3)-3' (SEQ ID NO: 14)

[0166] Those skilled in the art will recognize that PNA sequences containing appropriate reactive groups can form click adducts with another molecule that is also appropriately functionalized to undergo a "click" reaction. Indeed, those skilled in the art will recognize that in order for one member of a pair of click conjugates to react with the other member of the pair, thereby forming a covalent bond, the two members of a pair of click conjugates must have reactive functional groups capable of reacting with each other. The following table illustrates different pairs of reactive functional groups that react with each other to form a covalent bond. TIFF0007727385000017.tif140170

[0167] In some embodiments, groups present on an antibody (e.g., a primary or secondary antibody) are reduced in the presence of dithiothreitol ("DTT") to provide an antibody with one or more thiol groups. The thiol groups can be reacted with a first member of a click pair, which has a reactive functional group (e.g., a DBCO group) capable of participating in a "click chemistry" reaction. The first member of the click conjugate pair can also contain a second functional group (e.g., maleimide) capable of reacting with a thiolated antibody. The second functional group on the first member of the click conjugate pair is one that is unable to react in a click chemistry reaction. As shown in Figure 18, this step allows the antibody to be functionalized with a first reactive functional group capable of participating in a "click chemistry" linkage. The second member of the click pair, such as a PNA molecule, is then introduced, which contains a second reactive functional group (e.g., an azide group) capable of participating in a "click chemistry" reaction. The second member of the click pair can also contain a label or reporter moiety. In the embodiment shown in Figure 18, an antibody bearing a DBCO group can be linked to the azide group of the second member of the click member pair such that the PNA conjugate is linked to the antibody. PNAs conjugated by this procedure are neither chemically cleavable nor photocleavable.

[0168] In some embodiments, PNA sequences can be introduced to specific binding moieties and / or linkers using "maleimide" chemistry (see Figure 20). In some embodiments, PNAs conjugated by this procedure are not chemically cleavable or photocleavable.

[0169] Non-limiting examples of PNA sequences with SMCC groups are provided below: PNA 8: 5'-Biotin-O-GTCAACCATCTTCAG-Lys(SMCC)-3' (SEQ ID NO: 15)

[0170] Without wishing to be bound by any particular theory, it is believed that the use of click chemistry or maleimide chemistry allows for the introduction of shorter PNA sequences, for example, sequences having 10 or fewer bases.

[0171] Conjugate detection In some embodiments, the conjugate of any of formulas (IA), (IB), (IIA), (IIB), and (IIC) may include a label that facilitates direct detection of the conjugate. For example, if the label of the conjugate includes a fluorophore or chromophore, the fluorophore or chromophore can be directly detected according to methods known to those skilled in the art.

[0172] In other embodiments, specific reagents are utilized to enable detection of a conjugate of any of formulas (IA), (IB), (IIA), (IIB), and (IIC), thus enabling detection of the target in a tissue sample. In some embodiments, as further described herein, a detection reagent is utilized that is specific to a particular label of the conjugate or complementary to the nucleotide sequence (e.g., a PNA sequence) of the conjugate. In some embodiments, the detection reagent comprises a secondary antibody specific to the label of the conjugate; for example, the secondary antibody may be an anti-label antibody comprising a label (i.e., "X" in formulas (IA), (IB), (IIA), (IIB), and (IIC) herein). In some embodiments, the secondary antibody is an anti-hapten antibody, and the label is a hapten.

[0173] In some embodiments, secondary antibodies or anti-labeled antibodies can be conjugated to a "reporter moiety" to achieve detection of the conjugates of Formulae (I), (IA), (IC), (ID), and (II). In some embodiments, reporter moieties of secondary antibodies include chromogenic, fluorescent, phosphorescent, and luminescent molecules and materials, catalysts (e.g., enzymes) that convert one substance to another resulting in a detectable difference (e.g., by converting a colorless substance to a colored substance or vice versa, or by the formation of a precipitate or an increase in sample turbidity), haptens that can be detected through an antibody-hapten binding interaction using an additional detectably labeled antibody conjugate, and paramagnetic and magnetic molecules or materials. Of course, the reporter moiety itself can also be indirectly detected; for example, if the reporter moiety is a hapten, an additional antibody specific for that reporter moiety can be utilized to detect the reporter moiety, as known to those skilled in the art.

[0174] In some embodiments, the anti-labeled antibody is selected from the group consisting of DAB; AEC; CN; BCIP / NBT; Fast Red; Fast Blue; Fuchsin; NBT; ALK GOLD; Cascade Blue acetyl azide; Dapoxyl sulfonic acid / carboxylic acid succinimidyl ester; DY-405; Alexa Fluor 405 succinimidyl ester; Cascade Yellow succinimidyl ester; Pyridyloxazole succinimidyl ester (PyMPO); Pacific Blue succinimidyl ester; DY-415; 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester; DYQ-425; 6-FAM phosphoramidite; Lucifer Yellow; iodoacetamide; Alexa Fluor 430 succinimidyl ester; Dabcyl succinimidyl ester; NBD chloride / fluoride; QSY 35 succinimidyl ester; DY-485XL; Cy2 succinimidyl ester; DY-490; Oregon Green 488 carboxylic acid succinimidyl ester; Alexa Fluor 488 succinimidyl ester; BODIPY 493 / 503 C3 succinimidyl ester; DY-480XL; BODIPY FL C3 succinimidyl ester; BODIPY FL C5 succinimidyl ester; BODIPY FL-X succinimidyl ester; DYQ-505; Oregon Green 514 carboxylic acid succinimidyl ester; DY-510XL; DY-481XL; 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester (JOE); DY-520XL; DY-521XL; BODIPY R6G C3 succinimidyl ester; erythrosine isothiocyanate; 5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl ester; Alexa Fluor 532 succinimidyl ester; 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein succinimidyl ester (HEX); BODIPY 530 / 550 C3 succinimidyl ester; DY-530; BODIPY TMR-X succinimidyl ester; DY-555; DYQ-1; DY-556; Cy3 succinimidyl ester; DY-547;DY-549; DY-550; Alexa Fluor 555 succinimidyl ester; Alexa Fluor 546 succinimidyl ester; DY-548; BODIPY 558 / 568 C3 succinimidyl ester; Rhodamine Red-X succinimidyl ester; QSY7 succinimidyl ester; BODIPY 564 / 570 C3 succinimidyl ester; BODIPY 576 / 589 C3 succinimidyl ester; Carboxy-X-rhodamine (ROX); succinimidyl ester; Alexa Fluor 568 succinimidyl ester; DY-590; BODIPY 581 / 591 C3 succinimidyl ester; DY-591; BODIPY TR-X succinimidyl ester; Alexa Fluor 594 succinimidyl ester; DY-594; Carboxynaphthofluorescein succinimidyl ester; DY-605; DY-610; Alexa Fluor 610 succinimidyl ester; DY-615; BODIPY 630 / 650-X succinimidyl ester; Eriograsin; Alexa Fluor 633 succinimidyl ester; Alexa Fluor 635 succinimidyl ester; DY-634; DY-630; DY-631; DY-632; DY-633; DYQ-2; DY-636; BODIPY 650 / 665-X succinimidyl ester; DY-635; Cy5 succinimidyl ester; Alexa Fluor 647 succinimidyl ester; DY-647; DY-648; DY-650; DY-654; DY-652; DY-649; DY-651; DYQ-660; DYQ-661; Alexa Fluor 660 succinimidyl ester; Cy5.5 succinimidyl ester; DY-677; DY-675; DY-676; DY-678; Alexa Fluor 680 succinimidyl ester; DY-679; DY-680; DY-682; DY-681; DYQ-3; DYQ-700; Alexa Fluor 700 succinimidyl ester; DY-703; DY-701; DY-704; DY-700; DY-730; DY-731; DY-732; DY-734; DY-750; Cy7 succinimidyl ester; DY-749; DYQ-4;and a reporter moiety selected from the group consisting of Cy7.5 succinimidyl ester;

[0175] Fluorophores belong to several general 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, squaraine derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, and tetrapyrrole derivatives. In other embodiments, the fluorescent moiety is selected from 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, Phycobilisome) (Columbia Biosciences), and APC, APCXL, RPE, BPE (available from Phyco-Biotech, Greensea, Prozyme, Flogen).

[0176] In other embodiments, the anti-label antibody is conjugated to an enzyme. In some embodiments, suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase, or β-lactamase. In other embodiments, the enzyme includes an oxidoreductase or peroxidase (e.g., HRP, AP). In these embodiments, the enzyme conjugated to the anti-label antibody catalyzes the conversion of a chromogenic substrate into a reactive moiety that is covalently bound to a sample proximal to the target or directly on the target. Specific non-limiting examples of chromogenic compounds / substrates include diaminobenzidine (DAB), 4-nitrophenyl phosphate (pNPP), Fast Red, bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), BCIP / NBT, Fast Red, AP Orange, AP Blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline sulfonic acid] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β ... These include lactopyranoside (X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal), p-nitrophenyl-α-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue, tetrazolium violet, N,N'-biscarboxypentyl-5,5'-disulfonato-indo-dicarbocyanine (Cy5), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (DISCO Purple), and rhodamine 110. DAB, when oxidized in the presence of peroxidase and hydrogen peroxide, results in the deposition of a brown, alcohol-insoluble precipitate at the enzyme active site.

[0177] In some embodiments, the chromogenic substrate is a signaling conjugate comprising a latent reactive moiety and a chromogenic moiety. In some embodiments, the latent reactive moiety of the signaling conjugate is configured to undergo catalytic activation to form a reactive species that can covalently bond to a sample or other detection component. The catalytic activation is driven by one or more enzymes (e.g., oxidoreductase enzymes and peroxidase enzymes such as horseradish peroxidase) and results in the formation of reactive species. These reactive species can react with the chromogenic moiety in the vicinity of their generation, i.e., near the enzyme. Specific examples of signaling conjugates are disclosed in U.S. Patent Application Publication No. 2013 / 0260379, the disclosure of which is incorporated herein by reference in its entirety.

[0178] In embodiments in which the label is biotin, the PNA conjugate can be contacted with streptavidin linked to an enzyme (e.g., alkaline phosphatase or horseradish peroxidase). Without wishing to be bound by any particular theory, it is believed that streptavidin-AP or streptavidin-HRP conjugates specifically and irreversibly bind to the biotin-labeled PNA conjugate. The PNA-biotin-streptavidin conjugate can then be visualized using a chromogenic substrate for alkaline phosphatase or horseradish peroxidase (such as those described above) to generate a detectable signal. Similarly, if the label is biotin, the PNA conjugate can alternatively be contacted with streptavidin linked to a fluorophore (e.g., FTIC), and the signal for the fluorophore can be detected.

[0179] In embodiments in which the PNA tag is cleaved from the antibody conjugate (via a cleavable linker as described herein), the PNA sequence cleaved from the conjugate can be detected according to the methods described in "Peptide nucleic acid characterization by MALDI-TOF mass spectrometry," Anal Chem. 1996 Sep. 15;68(18):3283-7, the disclosure of which is incorporated herein by reference in its entirety. Similarly, the PNA sequence of a PNA conjugate can similarly be detected by other mass spectrometry methods, such as electrospray ionization (ESI), according to methods known to those of skill in the art.

[0180] Detection of conjugates using complementary nucleotide sequences, including complementary PNA or DNA sequences In some embodiments, the conjugates of formula (IA), (IB), (IIA), (IIB), and (IIC) can be detected by hybridizing one of a PNA sequence, a gamma PNA sequence, or a DNA sequence to the nucleotide sequence of the conjugate, wherein the PNA sequence or DNA sequence is complementary to the nucleotide sequence of the conjugate. For example, a PNA or DNA sequence complementary to the PNA sequence of the PNA conjugate can be detected after its hybridization to the PNA sequence of the PNA conjugate.

[0181] In some embodiments, the conjugates of formulas (IA), (IB), (IIA), (IIB), and (IIC) do not contain a label. In some embodiments, the PNA or DNA sequence complementary to the nucleotide sequence of the conjugate contains a reporter moiety, such as those described herein. In some embodiments, the reporter moiety is a chromogen. In other embodiments, the reporter moiety is a fluorophore (see, e.g., Figure 8E). In still other embodiments, the reporter moiety is a hapten (e.g., digoxigenin, as in Figure 9D). In further embodiments, the reporter moiety is an enzyme. In further embodiments, the reporter moiety is a nanoparticle (e.g., a gold nanoparticle that can be used in scanning electron imaging or quantum dots). Of course, in the case of gamma PNA sequences, multiple reporter moieties can be incorporated into the conjugate, as described herein and shown in Figure 17.

[0182] Of course, those skilled in the art will recognize that the same conjugate of formula (IA), (IB), (IIA), (IIB), and (IIC) can be used to provide multiple imaging modalities. For example, if a fluorescently labeled complementary DNA or PNA sequence is provided, it can be used for fluorescent imaging. Using the same conjugate of formula (IA), (IB), (IIA), (IIB), and (IIC), haptenized DNA or PNA sequence can also be used for effective chromogenic imaging.

[0183] Conjugate detection and / or quantification using the NanoString nCounter platform In some embodiments, the conjugates of formulas (IA), (IB), (IIA), (IIB), and (IIC) comprise oligomers having nucleotide sequences that can act as molecular "barcodes." For example, two PNA conjugates may contain similar PNA oligomer portions, but the PNA oligomer portions may differ at certain bases within the PNA sequence (e.g., even by a single change in one nucleotide). In this manner, PNA conjugates with different PNA sequences can be detected and / or quantified, such as by using the Nanostring nCounter platform. In some embodiments, the PNA conjugates comprise a reporter moiety, such as a biotin label.

[0184] In some embodiments, the conjugate of any of formulas (IA), (IB), (IIA), (IIB), and (IIC) comprises a cleavable linker. After introducing the conjugate of any of formulas (IA), (IB), (IIA), (IIB), and (IIC) into a sample, chemical reagents, enzymes, and / or radiation (e.g., UV, IR, etc.) are introduced into the sample to cleave the cleavable linker group, thereby releasing the nucleotide sequence of the conjugate (e.g., PNA-antibody sequence or gamma PNA sequence). This can, of course, be repeated for different conjugates of any of formulas (IA), (IB), (IIA), (IIB), and (IIC). Once all nucleotide sequences (e.g., PNA sequence or gamma PNA sequence) have been released, they can be detected and quantified as described herein. Those skilled in the art will also recognize that different conjugates can contain different cleavable linkers, thereby allowing different nucleotide sequences to be released at different times after introduction of different reagents / radiation, thereby enabling gradual detection and / or quantification. In some embodiments, the conjugate of any of formulas (IA), (IB), (IIA), (IIB), and (IIC) is a PNA conjugate, i.e., the conjugate comprises an oligomer comprising a PNA sequence or a gamma PNA sequence.

[0185] For example, if both PD-L1 and Ki67 markers are present on the same tissue section, an anti-PD-L1 / PNA1 conjugate and an anti-Ki67 PNA2 conjugate can be used to stain the tissue section. The PNA1 and PNA2 oligomer moieties may contain two different PNA sequences, but both are still chemically cleavable by a cleavable moiety within the conjugate, as described herein. After incubating the tissue with the two conjugate antibodies and carefully rinsing to remove unbound PNA-conjugated antibodies, the two different PNAs are cleaved from the conjugate. The two different PNAs can be counted using an nCounter (NanoString Technology) to determine the number of cleaved PNAs. The difference between the PNA1 and PNA2 counts reflects the difference in protein expression levels of the PD-L1 and Ki67 markers.

[0186] In these embodiments, because the detection scheme is based on hybridization of the reporter strand to a target oligomer (which can be DNA or PNA), the PNA sequence of the PNA conjugate can be detected and / or quantified in a manner similar to DNA. However, PNA conjugates are shorter than the standard DNA targets (approximately 70-100 bases long) typically detected by the NanoString nCounter platform. However, the presence of biotin on the 3' end of the PNA eliminates the need for a capture strand. Furthermore, the higher binding affinity of PNA to DNA compared to DNA to DNA provides sufficient stability for the relatively short PNA / DNA reporter duplex. The PNA sequence is mixed with a reporter strand that is designed to be complementary to the PNA sequence. After removing unbound PNA, the PNA / reporter construct is incubated on a streptavidin-coated cartridge and then aligned using an electric field. The nCounter is then used to read and count the reporter strands. The same procedure can be performed for multiple PNA sequences.

[0187] Detection and / or quantification of conjugates using Gyros Gyros is an immunoassay platform using an affinity flow-through format with parallel processing and laser-induced fluorescence detection. Assays are performed in a compact disc (CD) containing channels on the scale of over 100 nanoliters, using centrifugal force and capillary action for fluid delivery and movement.

[0188] In a typical, non-limiting experiment, a biotinylated epitope peptide is first bound to a 15 nL affinity capture column consisting of streptavidin-coated beads. After rinsing, a primary antibody specific to the epitope peptide flows through the column and binds to the peptide. A secondary antibody labeled with a fluorescent dye (e.g., AlexaFluor 647) then binds to the primary antibody, which is then detected and quantified using laser-induced fluorescence. To detect the oligomers of the present disclosure, a biotinylated PNA oligomer (see Figure 3D) is hybridized to a complementary single-stranded DNA conjugated to a reporter moiety (e.g., a hapten, including, but not limited to, digoxigenin). The biotin in the hybridized nucleic acid strand binds to the streptavidin-coated beads within the Gyros CD. The DIG label at the other end of the hybrid is then detected with an Ms-anti-DIG antibody followed by a goat anti-mouse antibody (GAM) labeled with AlexaFluor 647, facilitating quantitative measurement of the original oligomer. The principle of using Gyros technology for quantification is shown in Figure 32. Additional information regarding Gyros technology devices and methods of use is described in U.S. Patent Nos. 8,133,438 and 8,592,219, the disclosures of which are incorporated herein by reference in their entireties. Additional information regarding Gyros technology devices and methods of use is also described in U.S. Patent Application Publication Nos. 2011 / 0116972, 2011 / 0195524, and 2007 / 0241061, the disclosures of which are incorporated herein by reference in their entireties.

[0189] Any conjugate of formula (IA), (IB), (IIA), (IIB), and (IIC) can be used for quantification using the Gyros platform, provided that the conjugate contains a cleavable linker (e.g., a linker containing a disulfide group). After introducing a conjugate of formula (IA), (IB), (IIA), (IIB), and (IIC) into a sample, chemical reagents, enzymes, and / or radiation are introduced into the sample to cleave the cleavable linker group, thereby releasing the nucleotide sequence of the conjugate (e.g., a PNA sequence or a gamma PNA sequence). Quantification can then proceed as described above.

[0190] In some embodiments, the conjugate of any of formulas (IA), (IB), (IIA), (IIB), and (IIC) is a PNA conjugate, i.e., the conjugate comprises an oligomer comprising a PNA sequence or a gamma PNA sequence, and the PNA conjugate comprises a primary antibody conjugated to a PNA oligomer as described herein. In some embodiments, the introduced single-stranded DNA is complementary to the PNA sequence of the PNA conjugate and is capable of hybridizing with the PNA sequence. In some embodiments, the complementary single-stranded DNA sequence is conjugated to a reporter moiety. In some embodiments, the complementary single-stranded DNA sequence is conjugated to a hapten. In some embodiments, the complementary single-stranded DNA sequence is conjugated to digoxigenin.

[0191] In some embodiments, multiple different PNA conjugates can be introduced simultaneously or sequentially. Those skilled in the art will also recognize that different PNA conjugates may contain different cleavable moieties, and thus different PNA sequences may be released at different times after introduction of different reagents / radiation, thereby allowing for stepwise quantification.

[0192] In some embodiments, after quantification using the Gyros platform, the tissue is stained according to methods commonly used in the art. For example, after cleaving the PNA sequence or gamma PNA sequence from the PNA conjugate, the tissue to which the PNA conjugate is bound can be stained by introducing an anti-primary antibody containing a reporter moiety, as shown in Figure 33. In this way, quantification can be combined with the visualization of the target in the biological sample.

[0193] Detection kits containing PNA conjugates and detection reagents for detecting PNA conjugates In some embodiments, the conjugates of formula (IA), (IB), (IIA), (IIB), and (IIC) can be utilized as part of a "detection kit." Generally, any detection kit can include one or more conjugates of formula (IA), (IB), (IIA), (IIB), and (IIC), and detection reagents for detecting the one or more conjugates.

[0194] In some embodiments, a detection kit may include a first composition comprising a conjugate of any of formulas (IA), (IB), (IIA), (IIB), and (IIC), and a second composition comprising a detection reagent specific to the first composition, such that the conjugate can be detected via the detection kit. In some embodiments, a detection kit includes multiple conjugates of formulas (IA), (IB), (IIA), (IIB), and (IIC) (e.g., mixed together in a buffer or provided in individual shipping containers or compartments), wherein the detection kit also includes a detection reagent specific for each of the multiple conjugates.

[0195] For example, a kit can include a first PNA conjugate specific for a first target, a PNA conjugate having a first PNA oligomer portion, and a second PNA conjugate specific for a second target having a second PNA oligomer portion, where at least a portion of the first and second PNA oligomers are different. The kit may further include detection reagents specific for each of the different PNA conjugates.

[0196] As another example, the kit can include a first PNA conjugate having a first PNA oligomer portion (and no label), and the kit can further include a PNA or DNA sequence complementary to the PNA sequence of the first PNA oligomer portion.

[0197] As yet another example, a kit can include a series of different PNA conjugates, each specific for a different target and having a different PNA oligomer moiety, and each different PNA conjugate of the kit can serve as a different molecular "barcode" that can be used for qualitative and / or quantitative analysis.

[0198] Of course, any kit may contain other reagents, including buffers, counterstains, enzyme inactivation compositions, deparaffinization solutions, etc., if necessary for manual or automated target detection. The detection kit may also contain other specific binders (e.g., nucleic acid probes for ISH; unmodified (natural) antibodies, and antibody conjugates) and detection reagents for detecting these other specific binders. For example, the kit may contain one or more PNA conjugates; one or more anti-labeled antibodies for detecting one or more PNA conjugates; at least one unmodified antibody (i.e., a natural antibody not linked to a PNA sequence); and a detection reagent for detecting the at least one unmodified antibody. In some embodiments, instructions are provided for using the PNA conjugates and other components of the kit for use in an assay, such as an MIHC assay.

[0199] Methods for detecting targets using conjugates of any of formulae (IA), (IB), (IIA), (IIB), and (IIC), and detection reagents The present disclosure also provides methods for detecting one or more targets in a tissue sample using any of the conjugates of formula (IA), (IB), (IIA), (IIB), and (IIC) described herein. In some embodiments, a conjugate of any of formulas (IA), (IB), (IIA), (IIB), and (IIC) can be used in a simplex assay to directly or indirectly detect a specific target in a tissue sample (e.g., CD68, Ki67, CD20, etc.).

[0200] In some embodiments, a conjugate of any of Formulas (IA), (IB), (IIA), (IIB), and (IIC) comprises a primary antibody (e.g., an antibody specific for CD68, Ki67, CD20, etc.). In these embodiments, a conjugate comprising a primary antibody can be used to directly "label" a target with the conjugate. In other embodiments, a conjugate of any of Formulas (IA), (IB), (IIA), (IIB), and (IIC) comprises a secondary antibody. In these embodiments, as discussed in more detail herein, a target (e.g., a protein target or a nucleic acid target) can be labeled with a primary antibody (for IHC) or a nucleic acid conjugate (e.g., a nucleic acid sequence linked to a hapten in ISH), and the primary antibody or nucleic acid conjugate can then be "labeled" with a conjugate comprising a secondary antibody. These and other embodiments are further described herein.

[0201] In some embodiments, the PNA conjugate comprises a primary antibody if the PNA-primary antibody conjugate is specific for the target of interest, and if a target-PNA-primary antibody conjugate complex is formed upon application of the PNA-primary antibody conjugate to a tissue sample (see, e.g., Figures 22A-22D and 26). Following application of the PNA-primary antibody conjugate, a detection reagent (e.g., an anti-label antibody) can then be applied so that the target-PNA-primary antibody conjugate complex can be detected. In some embodiments, the detection reagent comprises an anti-label antibody specific for a particular label on the PNA-primary antibody conjugate, where the anti-label antibody comprises a reporter moiety. The single target can then be visualized or otherwise detected.

[0202] In other embodiments, a tissue sample is first contacted with a primary antibody or nucleic acid probe to form either a target-primary antibody complex or a target-nucleic acid probe complex. Next, a PNA conjugate containing a secondary antibody is introduced into the tissue sample, and the secondary antibody portion of the PNA conjugate is specific for either (i) the primary antibody, (ii) a label conjugated to the primary antibody, or (iii) a label conjugated to the nucleic acid probe. Application of the PNA-secondary antibody conjugate allows the formation of a secondary complex, allowing the target to be "labeled." After application of the PNA-secondary antibody conjugate and formation of the secondary complex, a detection reagent (e.g., an anti-label antibody) can be applied to detect the secondary complex. In some embodiments, the detection reagent includes an anti-label antibody specific to a particular label of the PNA-secondary antibody conjugate, where the anti-label antibody includes a reporter moiety. The target can then be visualized or otherwise detected.

[0203] In yet other embodiments, the tissue sample is first contacted with a PNA-primary antibody conjugate; or first with a primary antibody, followed by the introduction of a PNA-secondary antibody conjugate. After the introduction of each PNA conjugate, the sample can be contacted with a DNA or PNA sequence complementary to the PNA sequence of the PNA conjugate, where the complementary DNA or PNA sequence contains one or more reporter moieties (e.g., a chromogen, a fluorophore, an enzyme, or a hapten). In embodiments where the complementary DNA or PNA sequence contains a chromogen or a fluorophore, the "labeled" target complex can be directly detected. On the other hand, if the complementary DNA or PNA sequence contains a hapten, an anti-hapten antibody conjugated to a reporter moiety must be introduced to facilitate the final detection of the "labeled" target complex.

[0204] Of course, in alternative embodiments, the DNA or PNA sequence of any of the conjugates of formulas (IA), (IB), (IIA), (IIB), and (IIC) can be quantified after cleavage of the DNA or PNA sequence from the conjugate, using techniques such as the NanoString nCounter method or Gyros technology described herein. In some embodiments, the conjugate comprises a cleavable group and a biotin label. These methods would not require the use of any additional detection reagents.

[0205] In some embodiments of the present disclosure, methods for multiplexed detection, including automated multiplexed detection, are provided. Figure 14A provides a flowchart illustrating one method for multiplexed target detection, in which a tissue sample is simultaneously contacted with multiple PNA conjugates (step 100), each PNA conjugate specific to a particular target, and each PNA conjugate contains a different PNA oligomer (i.e., a PNA oligomer with a different PNA sequence and / or a different label). While Figure 14A illustrates the application of PNA conjugates, those skilled in the art will understand that PNA conjugates can include PNA-nucleic acid conjugates, PNA-primary antibody conjugates, and PNA-secondary antibody conjugates, depending on the target in the sample (e.g., a nucleic acid sequence recognized by a primary antibody PNA conjugate, a protein target, or a pre-deposited primary antibody recognized by a secondary antibody PNA conjugate). Of course, any PNA conjugate can have a PNA sequence with any number of nucleotides as described herein. Similarly, any PNA conjugate can have a PNA sequence that includes at least one nucleotide with a substitution at a gamma position, ie, a gamma PNA.

[0206] In some embodiments, the sample can be contacted with two PNA conjugates, where each PNA conjugate is specific to a particular target and each PNA conjugate comprises a different PNA oligomer moiety. In other embodiments, the sample can be contacted with three PNA conjugates, where each PNA conjugate is specific to a particular target and each PNA conjugate comprises a different PNA oligomer moiety.

[0207] PNA conjugates can be supplied to tissue samples as a "pool" or "cocktail" containing each PNA conjugate required for a particular assay. Pooling of PNA conjugates is considered possible because PNA conjugates are not expected to cross-react with each other, at least to the extent that any cross-reactivity does not interfere with staining performance. Each PNA conjugate will bind to its respective target and form a detectable target-PNA conjugate complex. In some embodiments, a blocking step is performed after applying the PNA conjugates.

[0208] Following simultaneous application of the PNA conjugates (step 100), multiple detection reagents are simultaneously applied to the tissue sample (step 110), where each detection reagent facilitates detection of one of the initially applied PNA conjugates (step 100) and each detection reagent contains a different reporter moiety. In some embodiments, the detection reagent is streptavidin conjugated to a fluorophore, chromophore, or hapten. In other embodiments, the detection reagent is a secondary antibody specific to the label of the PNA conjugate (e.g., an anti-hapten antibody specific to the hapten of the PNA conjugate). In still other embodiments, the detection reagent is a DNA or PNA sequence complementary to the PNA sequence of the PNA oligomer portion of the PNA conjugate. In embodiments in which anti-label antibodies are used, the anti-label antibodies can be provided to the tissue sample as a pool or cocktail containing each of the anti-label antibodies necessary for detection of the target-PNA conjugate complex. Following application of the detection reagents, in some embodiments, the tissue sample can be stained with a counterstain. The signal from each of the label and / or reporter moieties can be visualized or otherwise detected (eg, visualized or detected simultaneously).

[0209] An example of a multiplex assay utilizing PNA conjugates is as follows: A first PNA-antibody conjugate comprising a first PNA oligomer and specific for a first target (e.g., specific for one of CD68, Ki67, CD20, etc.) is introduced into a tissue sample. In some embodiments, the first PNA-antibody conjugate forms a detectable first target-PNA-antibody conjugate complex. Concurrently, a second PNA-antibody conjugate comprising a second PNA oligomer and specific for a second target (e.g., another one, such as CD68, Ki67, CD20, etc.) is introduced into the sample to form a second target-PNA-antibody conjugate complex. Third, fourth, and nth additional PNA-antibody conjugates (forming "n" target-detection probe complexes) with different PNA sequences and / or labels against other targets can further be introduced simultaneously with the first and second PNA-antibody conjugates.

[0210] After deposition of the PNA-antibody conjugates, they can, of course, be detected directly or indirectly depending on their composition. In some embodiments, anti-label antibodies are introduced to enable detection of each target-PNA-antibody conjugate complex. In some embodiments, the anti-label antibodies are specific for different labels on the PNA conjugates, and each anti-label antibody is conjugated to a different reporter moiety. In some embodiments, the detectable reagent is an anti-label antibody, each conjugated to a fluorophore. In some embodiments, the first, second, and nth anti-label antibodies are introduced simultaneously, where each of the first, second, and nth detection reagents is specific for a different PNA-antibody conjugate, and each anti-label antibody is conjugated to a fluorophore. In other embodiments, the first, second, and nth anti-label antibodies are introduced sequentially, where each of the first, second, and nth detection reagents is specific for a different PNA-antibody conjugate, and each anti-label antibody is conjugated to an enzyme.

[0211] Alternatively, PNA-antibody conjugates can be detected by introducing a PNA or DNA sequence complementary to the PNA sequence of the PNA oligomer portion of the introduced PNA conjugate. Each complementary PNA or DNA sequence can contain a reporter moiety, as described herein, including an enzyme, a fluorophore, a hapten, or a nanoparticle. If the complementary PNA or DNA sequence contains a hapten, an additional detection reagent (e.g., an anti-hapten antibody conjugated to an enzyme or a fluorophore) can be introduced to facilitate detection of the complementary PNA or DNA sequence, and thus the target, in the sample.

[0212] In a further example of a multiplex assay according to the present disclosure, a first PNA-antibody conjugate specific for a first target (e.g., CD3, Ki67, PD-L1, or an immune cell marker) and having a first label is introduced into a tissue sample. In some embodiments, the first PNA-antibody conjugate forms a detectable first target-PNA-antibody conjugate complex. Simultaneously or subsequently, a second PNA-antibody conjugate specific for a second target (e.g., another of CD3, Ki67, or PD-L1) and having a second label is introduced into the sample to form a second target-PNA-antibody conjugate complex. Third, fourth, and nth additional PNA-antibody conjugates (forming "n" target-PNA-antibody conjugate complexes), each specific for a different target, can be further introduced sequentially or simultaneously with the first and / or second PNA-antibody conjugate, where the third, fourth, and nth PNA-antibody conjugates each further have a different label. After deposition, the PNA-antibody conjugates can be detected. In some embodiments, additional detection reagents are introduced to enable target detection, including those described herein (e.g., chromogenic detection reagents). In some embodiments, the first, second, and nth detection reagents are introduced sequentially, wherein each of the first, second, and nth detection reagents comprises: (i) a secondary antibody specific for each of the labels of the PNA-antibody conjugate, i.e., an anti-label antibody, where the secondary antibody is conjugated to an enzyme; and (ii) a chromogenic substrate, wherein each of the first, second, and nth chromogenic substrates is different. In other embodiments, the first, second, and nth detection reagents are introduced sequentially, wherein each of the first, second, and nth detection reagents comprises a PNA or DNA sequence complementary to the PNA sequence of each PNA oligomer portion of the PNA conjugate, and each complementary PNA or DNA sequence comprises a reporter moiety.

[0213] In a further example of a multiplex assay according to the present disclosure, a first primary antibody specific for a first target (e.g., CD3, Ki67, PD-L1, or an immune cell marker) is introduced into a tissue sample (the first primary antibody is not a conjugate of any of Formulas (I), (IA), (IC), (ID), and (II)). Next, a first secondary antibody-PNA conjugate specific for the first primary antibody or a label conjugated to the first primary antibody is introduced, the first secondary antibody-PNA conjugate comprising a first PNA oligomer having a first PNA sequence. In some embodiments, the first secondary antibody-PNA-antibody conjugate forms a detectable first target-secondary antibody-PNA-antibody conjugate complex. The first PNA sequence of the secondary antibody-PNA conjugate is then cleaved from the conjugate.

[0214] Next, a second primary antibody specific for a second target (e.g., another one of CD3, Ki67, and PD-L1) is introduced into the sample. Next, a second secondary antibody-PNA conjugate specific for the second primary antibody or label conjugated to the second primary antibody is introduced, where the second secondary antibody-PNA conjugate comprises a second PNA oligomer having a second PNA sequence. In some embodiments, the second secondary antibody-PNA-antibody conjugate forms a detectable second target-secondary antibody-PNA-antibody conjugate complex. The second PNA sequence of the secondary antibody-PNA conjugate is then cleaved from the conjugate. Those skilled in the art will recognize that any number of primary antibodies and secondary antibody-PNA conjugates can be introduced sequentially, followed by cleaving the PNA sequence from the PNA oligomer of each secondary antibody-PNA conjugate. Finally, all of the different PNA sequences can be measured, and the target can be quantified.

[0215] In yet other embodiments, the multiplex detection method comprises the steps of: (i) contacting the biological sample with a first PNA-antibody conjugate to form a first target antibody-PNA conjugate complex; (ii) contacting the biological sample with a first labeled conjugate, the first labeled conjugate comprising a first enzyme (wherein the first labeled conjugate is an anti-label antibody that specifically binds to the first PNA-antibody conjugate and is configured to label the target with the enzyme); (iii) contacting the biological sample with a first signaling conjugate comprising a first latently reactive moiety and a first chromogenic moiety (see, e.g., U.S. Patent Application No. 13 / 849,160, the disclosure of which is incorporated herein by reference for a description of signaling conjugates and their components); and (iv) inactivating the first enzyme, such as by contacting the sample with a first enzyme inactivation composition, to substantially inactivate or completely inactivate the first enzyme contained in the biological sample.

[0216] After the first enzyme is inactivated (optionally), the multiplex method further includes the steps of: (v) contacting the biological sample with a second PNA-antibody conjugate to form a second target-PNA-antibody conjugate complex; (vi) contacting the biological sample with a second labeled conjugate, the second labeled conjugate comprising a second enzyme (wherein the second labeled conjugate is an anti-label antibody configured to specifically bind to the second PNA-antibody conjugate and label the target with the enzyme); (vii) contacting the biological sample with a second signaling conjugate comprising a second latently reactive moiety and a second chromogenic moiety; and (viii) inactivating the second enzyme, such as by contacting the sample with a first enzyme inactivation composition, to substantially inactivate or completely inactivate the first enzyme contained in the biological sample.

[0217] After the second enzyme is inactivated, the method can be repeated to introduce additional PNA-antibody conjugates together with additional detection reagents to achieve detection of other targets. After introducing all PNA-antibody conjugates (and other detection probes) and their respective detection reagents or kits, the method can further include counterstaining the sample and / or detecting signals from the first, second, and nth chromogenic moieties (manually or by automated methods), where each of the first, second, and nth chromogenic moieties is different from the others. Alternatively, each of the PNA-antibody conjugates can be added simultaneously or sequentially, but before adding any labeled conjugates. As another example, three PNA-antibody conjugates can be applied sequentially first before introducing any detection reagents, and then each detection reagent can be added sequentially.

[0218] In the context of multiplex assays in which multiple targets are detected sequentially and detection employs the use of enzymes, it is desirable to inactivate any reagents or endogenous enzymes between successive detection steps. As a result, the enzymes present in any one detection step will not interfere with the enzymes present in subsequent detection steps. This, in turn, will improve the visualization and detection of different detectable moieties used in the multiplex assay. Any enzyme inactivation composition known in the art can be used for this purpose. In some embodiments, an enzyme inactivation composition is applied to inactivate reagents or endogenous enzymes after each detection step. Exemplary enzyme inactivation compositions are disclosed in co-pending U.S. patent application Ser. No. 62 / 159,297, the disclosure of which is incorporated herein by reference in its entirety.

[0219] In some embodiments, the denaturing step prevents the enzyme used in the first set of detection reagents from acting on the second substrate. In some embodiments, the denaturant is a substance that denatures the enzyme in the first set of detection reagents. In some embodiments, the denaturant is, for example, formamide, alkyl-substituted amide, urea or urea-based denaturant, thiourea, guanidine hydrochloride, or a derivative thereof. Examples of alkyl-substituted amides include, but are not limited to, N-propylformamide, N-butylformamide, N-isobutylformamide, and N,N-dipropylformamide. In some embodiments, the denaturant is provided in a buffer solution. For example, formamide is used in hybridization with 20 mM dextran sulfate (50-57% formamide (UltraPure formamide stock), 2x SSC (20x SSC stock containing 0.3 M citrate and 3 M NaCl), 2.5 mM EDTA (0.5 M EDTA stock), 5 mM Tris, pH 7.4 (1 mM Tris, pH 7.4 stock), 0.05% Brij-35 (10% stock containing polyoxyethylene (23) lauryl ether), pH 7.4). The denaturing agent may be provided in a denaturing buffer. In some embodiments, the sample is treated with a denaturing agent for a period and under conditions sufficient to denature the first target probe detection enzyme, e.g., alkaline phosphatase. In some embodiments, the sample is treated with the denaturing agent at about 37°C for about 15 to about 30 minutes, preferably about 20 to 24 minutes. In some embodiments, the sample is treated with the denaturing agent for a period and under conditions sufficient to denature the target enzyme while maintaining hybridization of the second nucleic acid probe to the target.

[0220] In these embodiments using an anti-labeled antibody conjugated to an enzyme, suitable conditions are used to introduce the signaling conjugate or chromogenic substrate along with the biological sample. These conditions typically include providing a reaction buffer or solution containing a peroxide (e.g., hydrogen peroxide) and having a suitable salt concentration and pH to allow or promote the enzyme to perform its desired function. Generally, this step of the method is carried out at a temperature ranging from about 35°C to about 40°C, although one of skill in the art can select an appropriate temperature range appropriate for the selected enzyme and signaling conjugate. For example, these conditions will allow the enzyme and peroxide to react and promote radical formation at the latent reactive moiety of the signaling conjugate. The latent reactive moiety, and therefore the signaling conjugate as a whole, is covalently deposited on the biological sample, particularly on one or more tyrosine residues in proximity to the immobilized enzyme conjugate, the tyrosine residues of the enzyme portion of the enzyme conjugate, and / or the tyrosine residues of the antibody portion of the enzyme conjugate. The biological sample can then be illuminated with light, and the target can be detected through the absorbance of light produced by the chromogenic moiety of the signaling conjugate.

[0221] Detection methods using conjugates of any of formulae (IA), (IB), (IIA), (IIB), and (IIC) in conjunction with other specific binding entities In some embodiments of the present disclosure, conjugates of any of formulas (IA), (IB), (IIA), (IIB), and (IIC) are used in conjunction with other specific binders to perform multiplexed detection of targets in tissue samples. Those skilled in the art will recognize that any of the above-identified methods and procedures can be adapted for any assay using both conjugates of any of formulas (IA), (IB), (IIA), (IIB), and (IIC) and other specific binders.

[0222] In some embodiments, other specific binders include nucleic acids for in situ hybridization and unmodified antibodies for IHC. As used herein, the term "unmodified antibody(ies)" refers to antibodies that do not contain a nucleotide sequence (e.g., a DNA, PNA, or gamma PNA nucleotide sequence identified herein), but include antibodies conjugated to a hapten or another label. Essentially, an "unmodified antibody" is a native antibody traditionally used in IHC assays, which is specific for a particular target (e.g., an anti-CD3 antibody) and can be detected with an anti-species secondary antibody or, if a label is included, an anti-label antibody, etc. As an example, a rabbit anti-CD3 antibody can be detected with a goat anti-rabbit antibody. Similarly, a rabbit anti-CD3 antibody conjugated to a hapten can be detected with an anti-hapten antibody.

[0223] 14B and 14C illustrate a method for multiplexed detection of targets in which a tissue sample is contacted (simultaneously or sequentially) with one or more unmodified primary antibodies (first step, 220), followed by contact (simultaneously or sequentially) with one or more PNA conjugates (second step, 250). Those skilled in the art will recognize that the first step 220 and the second step 250 can be reversed, with the PNA conjugates applied to the tissue sample first, followed by the unmodified antibodies. Those skilled in the art will also recognize that appropriate nucleic acid probes (including those conjugated to labels) may be used in place of the unmodified antibodies, such that the multiplexed assay includes both ISH and IHC steps or stages (in any order).

[0224] In some embodiments, as shown in Figure 14B, a first unmodified primary antibody can be applied to a tissue sample to form a first target-primary antibody complex (step 200). A first detection reagent specific for the unmodified primary antibody is then applied to the tissue sample to detect the first target-primary antibody complex (step 210). Dashed line 205 in Figure 14B indicates that steps 200 and 210 of the first stage 220 can be repeated one or more times to provide sequential multiplexed detection of multiple different targets within a tissue sample using unmodified primary antibodies. For example, a second unmodified primary antibody can be applied to the tissue sample to form a second target-primary antibody complex (200), and then a second detection reagent specific for the second unmodified primary antibody is applied to detect the second target-primary antibody complex (210).

[0225] Figure 14C depicts an alternative method for multiplexed detection of targets using a two-step method similar to that presented in Figure 14B. In the method shown in Figure 14C, in step 260, each of the unmodified antibody conjugates is simultaneously introduced into a tissue sample. Next, in step 270, the sample is contacted with a detection reagent (e.g., an anti-species antibody or an anti-labeled antibody) to achieve detection of the unmodified antibodies. In an alternative embodiment, all of the unmodified primary antibodies can be applied sequentially (step 260), followed by the sequential application of each anti-species antibody (or anti-hapten antibody, if appropriate) (step 270).

[0226] Those skilled in the art will recognize that the detection reagent for the unmodified antibody can include an anti-species antibody specific for the unmodified antibody utilized. Alternatively, the detection reagent for the unmodified antibody can include an anti-hapten antibody specific for the hapten conjugated to the unmodified antibody. Those skilled in the art will also recognize that the anti-species or anti-hapten antibody can include a reporter moiety, and in embodiments in which the reporter moiety is an enzyme, an additional chromogenic substrate can be provided along with the first and second detection reagents.

[0227] Following the first stage of the multiplex assay 220 (FIG. 14B or 14C), a second stage 250 is performed, in which the tissue sample is simultaneously or sequentially contacted with multiple PNA conjugates (step 230), each PNA conjugate specific to a particular target and each PNA conjugate containing a different PNA oligomer moiety. The PNA conjugates may be provided to the tissue sample as a "pool" or "cocktail" containing each PNA conjugate required for a particular assay. Each PNA conjugate forms a detectable target-PNA conjugate complex with its specific target. Following the simultaneous or sequential application of the PNA conjugates (step 230), anti-label antibodies (secondary antibodies) are simultaneously applied to the tissue sample (step 240), where each anti-label antibody is specific to one of the applied PNA conjugates and each anti-label antibody contains a different reporter moiety. The anti-labeled antibodies may be supplied to the tissue sample as a "pool" or "cocktail" containing each anti-labeled antibody required for detection of the target-PNA-antibody complex.

[0228] Alternatively, after introducing each PNA conjugate in step 230, the sample can be contacted in step 240 with a PNA sequence complementary to the PNA sequence of the PNA conjugate or a DNA sequence, where the DNA sequence contains a reporter moiety (e.g., an enzyme, chromogen, fluorophore, or hapten). In embodiments where the DNA sequence contains a chromogen or fluorophore, the "labeled" target complex can be detected directly. On the other hand, if the DNA sequence contains a hapten, an anti-hapten antibody conjugated to a reporter moiety must be introduced to facilitate eventual detection of the "labeled" target complex.

[0229] Of course, in an alternative embodiment, the PNA sequence of each PNA conjugate can be quantified using, for example, the NanoString nCounter methodology described herein.

[0230] In some embodiments, the tissue sample may be stained with a counterstain after step 250. The signal from each reporter moiety (e.g., from the anti-species antibody or anti-label antibody) can be visualized or otherwise detected (e.g., visualized or detected simultaneously).

[0231] As an example of a multiplex assay comprising both (i) unmodified antibodies and (ii) PNA-antibody conjugates according to the present disclosure, a first antibody conjugate (e.g., an anti-CD3 antibody accidentally indirectly conjugated with a hapten label) is introduced into a tissue sample to form a target-antibody-conjugate complex. Simultaneously, an unmodified antibody (e.g., a rabbit anti-PDL1 antibody) is introduced into the tissue sample to form a target-unmodified antibody complex. Detection reagents are then introduced (simultaneously or sequentially) to detect the formed target-antibody-conjugate complex (e.g., an anti-hapten antibody) and the formed target-unmodified antibody complex (e.g., a goat anti-rabbit antibody), each of which is conjugated to a different fluorophore.

[0232] In the second stage of the multiplex assay, a first PNA-antibody conjugate containing a first PNA oligomer and specific for a first target (e.g., specific for CD68) is introduced into the tissue sample. In some embodiments, the first PNA-antibody conjugate forms a detectable first target-PNA-antibody conjugate complex. Sequentially or simultaneously, a second PNA-antibody conjugate containing a second PNA oligomer and specific for a second target (e.g., specific for Ki67) is introduced into the sample to form a second target-PNA-antibody conjugate complex. Additional third, fourth, and nth PNA-antibody conjugates specific for other targets (forming "n" target-PNA-antibody complexes) and having different PNA oligomers can be further introduced simultaneously with the first and second PNA-antibody conjugates. In an alternative embodiment, PNA-antibody conjugates may be added sequentially, with the PNA sequence being cleaved from the PNA-antibody conjugate prior to the introduction of the next PNA-antibody conjugate, and the cleaved PNA sequences can then be measured together.

[0233] After deposition of the PNA-antibody conjugates, they can, of course, be detected directly or indirectly depending on their composition. In some embodiments, the first, second, and nth detection reagents are introduced simultaneously, where each of the first, second, and nth detection reagents is specific to a different PNA-antibody conjugate. In other embodiments, the first, second, and nth detection reagents are introduced sequentially, where each of the first, second, and nth detection reagents is specific to a different PNA-antibody conjugate. In some embodiments, anti-label antibodies are introduced to enable detection of each target-PNA-antibody conjugate complex. In some embodiments, the detection reagents are anti-label antibodies specific to different labels of the PNA-antibody conjugates, each of which is conjugated to a reporter moiety, such as a fluorophore or enzyme. In some embodiments, the detectable reagents are anti-label antibodies each of which is conjugated to a fluorophore. In other embodiments, the detectable reagents are anti-labeled antibodies, each conjugated to an enzyme. In yet other embodiments, the detectable reagents are a combination of an anti-labeled antibody conjugated to a fluorophore and an anti-labeled antibody conjugated to an enzyme. In these embodiments in which the anti-labeled antibody is conjugated to an enzyme, a substrate for the enzyme is provided for detection (as described above). In other embodiments, a PNA or DNA sequence complementary to one or more PNA sequences and comprising an enzyme, fluorophore, or hapten is introduced to enable detection of each target-PNA-antibody conjugate complex. In yet other embodiments, the detection reagent is a PNA or DNA sequence complementary to the PNA sequence of the PNA oligomer portion of the PNA conjugate. Those skilled in the art will recognize that when complementary PNA or DNA sequences conjugated to a hapten are provided, additional reagents can be provided in the sample to facilitate detection of the complementary PNA or DNA sequences.In an alternative embodiment, the PNA-conjugated antibody may be hybridized to a complementary PNA or DNA sequence ex situ, and the complex, i.e., the PNA-conjugated antibody hybridized to the complementary PNA or DNA sequence, can then be introduced into tissue to allow labeling of targets within the sample.

[0234] automation Multiplex assays and methods can be automated and combined with a sample processor. The sample processor can be an automated device, such as the BENCHMARK XT instrument or the SYMPHONY instrument sold by Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. is the assignee of numerous U.S. patents disclosing systems and methods for performing automated analyses, 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. Patent Application Publication Nos. 2003 / 0211630 and 2004 / 0052685, each of which is incorporated herein by reference in its entirety. Alternatively, samples can be processed manually.

[0235] The specimen processor can apply fixatives to the specimen, including crosslinkers (e.g., aldehydes such as formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde crosslinkers), oxidizing agents (e.g., metal ions and metal complexes such as osmium tetroxide and chromate), 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, methacin, Bouin's solution, B5 fixative, Rossmann's solution, and Gendre's solution), microwaves, and various other fixatives (e.g., excluded volume fixation and vapor fixation).

[0236] If the specimen is a paraffin-embedded sample, the specimen can be deparaffinized in a specimen processing device using an appropriate deparaffinization solution. After the deparaffinization solution is removed by a waste removal agent, any number of substances can be applied sequentially to the specimen. The substances can be for pretreatment (e.g., protein cross-linking, nucleic acid exposure, etc.), denaturation, hybridization, washing (e.g., stringent washing), detection (e.g., ligation of visual or marker molecules with probes), amplification (e.g., amplification of proteins, genes, etc.), counterstaining, coverslipping, etc.

[0237] The sample processor can apply a wide range of substances to the sample. Substances include, but are not limited to, dyes, probes, reagents, rinses, and / or conditioners. Substances can be fluids (e.g., gases, liquids, or gas / liquid mixtures). Fluids can be solvents (e.g., polar solvents, nonpolar solvents, etc.), solutions (e.g., aqueous or other types of solutions), etc. Reagents can include, but are not limited to, dyes, wetting agents, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.), antigen retrieval fluids (e.g., aqueous or non-aqueous antigen retrieval solutions, antigen retrieval buffers, etc.). Probes can be isolated nucleic acids or isolated synthetic oligonucleotides attached to a detectable label or reporter molecule. Labels can include radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.

[0238] After the specimen is processed, the user can transport the slide with the specimen to the imaging device. The imaging device used herein is a brightfield imager slide scanner. One brightfield imager is the iScan Coreo™ brightfield scanner sold by Ventana Medical Systems, Inc. In automated embodiments, the imaging device is a digital pathology device such as that disclosed in International Patent Application No. PCT / US2010 / 002772 (International Publication No. WO / 2011 / 049608) entitled "IMAGING SYSTEM AND TECHNIQUES," or in U.S. Patent Application Publication No. 2014 / 0178169, filed September 9, 2011, entitled "IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME."

[0239] Counterstaining Counterstaining is a method of post-processing a sample after it has already been stained with an agent to detect one or more targets so that those structures can be more easily visualized under a microscope. For example, counterstaining may be used before coverslipping to make immunohistochemical staining more apparent. Counterstains differ in color from the primary stain. Many counterstains are known, including hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian blue, and Nuclear Fast Red. DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye that can be used.

[0240] In some cases, two or more dyes can be mixed together to create a counterstain. This allows for flexibility and the ability to select a stain. For example, a first stain can be selected for a mixture that has a particular attribute but still lacks a different desired attribute. A second stain can be added to the mixture to exhibit the missing desired attribute. For example, toluidine blue, DAPI, and pontamine sky blue can be mixed together to form a counterstain.

[0241] Imaging Certain aspects or all of the disclosed embodiments can be automated and facilitated by computer analysis and / or image analysis systems. In some applications, precise color or fluorescence ratios are measured. In some embodiments, an optical microscope is utilized for image analysis. Certain disclosed embodiments involve digital image acquisition. This can be accomplished by connecting a digital camera to the microscope. Digital images obtained from stained samples are analyzed using image analysis software. Color or fluorescence can be measured in several different ways. For example, color can be measured as red, blue, and green values; as hue, saturation, and brightness values; and / or by measuring specific wavelengths or wavelength ranges using a spectral imaging camera. Samples can also be evaluated qualitatively and semi-quantitatively. Qualitative evaluations include assessing staining intensity, identifying positively stained cells and subcellular compartments involved in the staining, and assessing overall sample or slide quality. Separate evaluations are performed on test samples, and this analysis can include comparison to known average values ​​to determine whether the sample represents an abnormal condition.

[0242] Samples and targets The sample contains biological components and is generally suspected to contain one or more target molecules of interest. The target molecule may be present on the surface of a cell, or the cell may be present in a suspension or in a tissue section. The target molecule may also be present inside a cell and can be detected upon cell lysis or cell penetration by a probe. Those skilled in the art will understand that the method for detecting a target molecule in a sample will vary depending on the type of sample and probe used. Methods for collecting and preparing samples are known in the art.

[0243] Samples for use in method embodiments, such as tissues or other biological samples, having compositions disclosed herein, can be prepared by those skilled in the art using any method known in the art. Samples can be obtained from subjects for routine screening or from subjects suspected of having a disorder, such as a genetic abnormality, infection, or neoplasia. Described embodiments of the disclosed methods can also be applied to samples free of genetic abnormalities, diseases, disorders, etc., referred to as "normal" samples. Such normal samples are particularly useful as controls for comparison with other samples. Samples can be analyzed for many different purposes. For example, samples can be used in scientific research or for diagnosis of suspected illnesses, or as prognostic indicators for treatment success, survival, etc.

[0244] The sample may contain multiple targets to which the probe or reporter molecule can specifically bind. The target may be a nucleic acid sequence or a protein. In some cases, the target is a protein or nucleic acid molecule derived from a pathogen, such as a virus, a bacterium, or an intracellular parasite, such as a viral genome. For example, the target protein may be produced from a target nucleic acid sequence that is related to a disease (e.g., correlated with a disease, involved in the cause, etc.).

[0245] Those skilled in the art will recognize that PNA conjugates specific for any of the following targets can be developed:

[0246] 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., cancer). In particular, numerous chromosomal abnormalities (including translocations and other rearrangements, amplifications or deletions) have been identified in neoplastic cells such as cancer cells (e.g., B-cell and T-cell leukemia), lymphoma, breast cancer, colon cancer, and neural cancer. Thus, in some examples, 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 a subset of cells in the sample.

[0247] In another example, the target protein is produced from a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) that is a tumor suppressor gene that is deleted (lost) in malignant cells.For example, the p16 region (including D9S1749, D9S1747, p16(INK4A), p14(ARF), D9S1748, p15(INK4B) and D9S1752) located on chromosome 9p21 is deleted in certain bladder cancers. Chromosomal deletions involving the distal region of the short arm of chromosome 1 (e.g., encompassing SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322) and the pericentromeric region of chromosome 19 (e.g., encompassing MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, and GLTSCR1) are characteristic molecular features of certain types of solid tumors of the central nervous system.

[0248] Many other cytogenetic abnormalities correlated with neoplastic transformation and / or proliferation are known to those of skill in the art. Target proteins produced by nucleic acid sequences (e.g., genomic target nucleic acid sequences) that correlate with neoplastic transformation and are useful in the disclosed methods 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 (5 p15.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK™ Accession No. NC-000008, nucleotides 19841058 to 19869049), RB1 (13q14; e.g., GENBANK™ Accession No. NC-000013, nucleotides 47775912 to 47954023), p53 (17p13.1; e.g., GENBANK™ Accession No. NC-000017, complement, nucleotides 7512464 to 7531642). ), N-MYC (2p24; e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides 151835231 to 151854620), CHOP (12q13; e.g., GENBANK™ Accession No. NC-000012, complement, nucleotides 56196638 to 56200567), FUS (16p11.2; e.g., GENBANK™ Accession No. NC-000016, nucleotides 31098954 to 31110601), FKHR (13p1 4; e.g., GENBANK™ Accession No. NC-000013, complement, nucleotides 40027817-40138734), as well as, e.g., ALK (2p23; e.g., GENBANK™ Accession No. NC-000002, complement, 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, complement, nucleotides 58941559 to 59137593), BCL6 (3q27; e.g., GENBANK™ Accession No. NC-000003, complement, nucleotides 188921859 to 188946169), MALF1, AP1 (1p32-p31; e.g., GENBANK™ Accession No. NC-000001, complement, nucleotides 59019051 to 59022373), TOP2A (17q21-q22; e.g., GENBANK™ Accession No. GENBANK™ Accession No. NC-000017, complement, nucleotides 35798321 to 35827695), TMPRSS (21q22.3; e.g., GENBANK™ Accession No. NC-000021, complement, nucleotides 41758351 to 41801948), ERG (21q22.3; e.g., GENBANK™ Accession No. NC-000021, complement, nucleotides 38675671 to 38955488); ETV1 (7p21.3; e.g., GENBANK™ Accession No. NC-000007, complement , nucleotides 13897379 to 13995289), EWS (22q12.2; e.g., GENBANK™ Accession No. NC-000022, nucleotides 27994271 to 28026505); FLI1 (11q24.1-q24.3; e.g., GENBANK™ Accession No. NC-000011, nucleotides 128069199 to 128187521), PAX3 (2q35-q37; e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides 222772851 to 2228 71944), PAX7 (1p36.2-p36.12; e.g., GENBANK™ Accession No. NC-000001, nucleotides 18830087-18935219), PTEN (10q23.3; e.g., GENBANK™ Accession No. NC-000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g., GENBANK™ Accession No. NC-000019, complement, nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK™ Accession No. NC-000001, complement, nucleotides 40133685 to 40140274), REL (2p13-p12; e.g., GENBANK™ Accession No. NC-000002, nucleotides 60962256 to 61003682), and CSF1R (5q33-q35; e.g., GENBANK™ Accession No. NC-000005, complement, nucleotides 149413051 to 149473128). [Example]

[0249] Each non-limiting example provided herein incorporates the use of PNA conjugate.Applicant provides that the PNA conjugate disclosed herein is suitable for use in IHC assay.Of course, as detailed herein, PNA conjugate can be used in conjunction with other detectable specific binding entities, and can be utilized in the combined assay of IHC and ISH.

[0250] Example 1 PNA / antibody conjugation Exemplary conjugations of PNA to goat anti-mouse (GAM), goat anti-rabbit (GAR), and mouse anti-DIG are described below:

[0251] (1) 100 micrograms of antibody (0.667 nmoles) was diluted to 2 mg / mL in PBS and treated with 15 molar equivalents of SPDP-PEG-NHS (10 nmoles from a 10 mM DMSO stock solution) for 2 hours. SPDP-PEG-NHS was available from QuantaBiodesign, Ohio, USA (SPDP-dPEG8-NHS ester, product number 10376).

[0252] (2) The labeled antibody was purified using a 7KD MW cutoff Zeba spin desalting column from ThermoFisher (product number 89882).

[0253] (3) Two nanomoles of PNA (8 μL from a 251 μM PNA stock solution in 1:1 (v / v) water / DMF) was added to the antibody solution along with 5 μL of DMF and 35 μL of PBS, resulting in a total reaction volume of approximately 100 μL. The mixture was incubated overnight at room temperature.

[0254] (4) The conjugate was then purified using a Zeba spin desalting column, which was then used to purify the PNA-conjugated antibody from the reaction mixture.

[0255] The purified PNA-conjugated antibody was diluted in a suitable diluent before use.

[0256] Example 2 UV-Vis measurements of antibody-PNA conjugates The PNA-conjugated antibodies were characterized by UV-Vis absorbance. An increase in absorbance at 260 nm for the antibody-PNA conjugate indicated successful PNA incorporation (see Figures 2A and 2B). The ratio of A260 to A280 qualitatively assesses the efficiency of conjugation and can potentially be used to estimate the number of PNA oligomers per antibody. This is also shown in Figures 30 and 31.

[0257] Example 3 Detection of PNA-antibody conjugates using SA-HRP: To demonstrate that conjugation of PNA oligomers (15-nt PNA, 10-nt PNA, or 15-nt gamma PNA) does not affect the binding affinity and specificity of the antibodies, IHC assays were performed on tonsillar tissue for different markers using PNA-conjugated antibodies (primary and secondary). All IHC assays were performed on a BenchmarkXT (Ventana) using a protocol modified based on the specific assay. PNA-conjugated antibodies were added manually at the appropriate concentrations in a titration of 100 μL.

[0258] The biotin label on the PNA tag was detected. Two tonsillar slides were incubated with anti-CD45 and anti-Ki67 primary antibodies, followed by PNA-conjugated GAR and GAM secondary antibodies (PNA sequences with 15 bases), respectively. The slides were then incubated with streptavidin-HRP (SA-HRP), followed by DAB deposition (chromogenic detection). The markers were observed at their expected locations (Figures 3A and B). No background signal was observed when the primary antibody was omitted from the assay (Figure 3C). Tonsillar slides were also successfully stained against CK5 / 6 using a primary haptenized antibody (anti-CK5 / 6:DIG) and a secondary PNA-conjugated anti-hapten antibody, followed by SA-HRP and DAB deposition (Figures 4A and 4B). The same experiment was performed with a PNA sequence with 10 bases. Tonsil tissue was stained with anti-Ki67, and then GAR conjugated to PNA sequence has 10 bases. The terminal biotin moiety of the PNA sequence with 10 bases conjugated to GAR was detected using SA-HRP followed by DAB deposition (Figure 21A). For comparison, tonsil tissue was stained for Ki67 with GAR-HRP (conventional Ultraview detection by Ventana). This experiment showed that the functionality of GAR was not changed by 10-base PNA sequence conjugation.

[0259] Tonsils were stained with anti-Ki67, followed by GAR (15 bases) conjugated to a gamma PNA sequence. The biotin at the end of the gamma PNA sequence was detected by SA-HRP followed by DAB deposition (Figure 28). The figure shows that gamma PNA can be successfully conjugated to a secondary antibody without any mislocalization.

[0260] These first experiments provide strong evidence that various PNA sequence lengths (10 and 15 bases) and properties (conventional PNA and gamma PNA) can be efficiently conjugated to secondary antibodies and that conjugation does not affect their binding affinity and specificity for the corresponding primary antibodies on tonsil slides.

[0261] PNA tags (10-base PNA and 15-base gamma PNA) were directly conjugated to primary antibodies and detected via the biotin moiety on the PNA sequence, as previously described. Tonsil slides were incubated with anti-CD3, anti-CD8, anti-CD34, and anti-Ki67 primary antibodies conjugated to a 10-base PNA sequence (designated sPNA1) with biotin at its terminus. The slides were then incubated with streptavidin-HRP (SA-HRP), followed by DAB deposition (chromogenic detection). All markers were observed in their expected locations. Figures 22A, 22B, 22C, and 22D show tonsil slides stained with anti-CD3 conjugated to a 10-base PNA, anti-CD8 conjugated to a 10-base PNA, anti-CD34 conjugated to a 10-base PNA, and anti-Ki67 conjugated to a 10-base PNA, respectively.

[0262] Figures 23 and 24 show that tonsil slides were stained with anti-CD3 and anti-CD8, respectively, conjugated to various 10-base PNA sequences, designated sPNA2, which have biotin at their termini and were detected as described above.

[0263] Tonsil slides were also stained with anti-CD3, anti-CD8, and anti-PD-L1 conjugated to 15-base gamma PNA sequences. The biotin moieties on the gamma PNA sequences were detected by incubation with SA-HRP followed by DAB deposition. Figures 29A, B, and C show tonsil slides stained with different concentrations of anti-CD3, anti-CD8, and anti-PD-L1, respectively.

[0264] Example 4A Chemical cleavage of PNA oligos In some embodiments, PNA-conjugated antibodies are designed to enable multiplexed quantitative measurement of protein expression using methods such as Gyros technology or the NanoString nCounter platform based on DNA counting. Therefore, the PNA sequence must be cleaved from the PNA-conjugated antibody after binding to tissue to enable ex situ PNA counting. In this particular example, the PNA sequence is bound to the antibody via a disulfide bond, which can be chemically cleaved (e.g., by reducing the disulfide bond) to release the PNA sequence.

[0265] Figures 5A-5D show tonsillar slides stained for Ki67 and CD45 using primary antibodies followed by PNA-conjugated anti-species secondary antibodies and detected by SA-HRP DAB deposition. Incubation of the slides with 20 mM TCEP (tris(2-carboxyethyl)phosphine, a reducing agent) prior to SA-HRP treatment resulted in complete loss of brown staining, indicating removal of the PNA tag.

[0266] Example 4B Photocleavage of PNA oligos Tonsil slides were treated with a primary antibody (rabbit Ki67) and a secondary antibody (goat anti-rabbit antibody with GAR-PL-PNA, a photocleavable PNA with biotin attached thereto). The slides were then irradiated with UV light (handheld UV lamp, 365 nm) for different periods of time. Control slides were not UV-treated and were used for comparison. All slides were then treated with SA-HRP and DAB for detection.

[0267] Figures 12A, 12B, and 12C show that the irradiated slides lacked color, indicating photocleavage of PNA sequences. In this experiment, the entire slide was irradiated with UV light. Cleavage of PNA from the entire slide can be achieved by chemical reduction of disulfide bonds. Photoirradiation offers the possibility of selectively irradiating specific areas of interest on the slide. To prove this concept, we used a laser capture microdissection (LCM) system to achieve selective irradiation. LCM uses UV light passing through an objective lens (high spatial precision) to cut specific areas of tissue (down to single cells). We used UV to selectively irradiate specific areas of tissue and photocleave PNA. Figure 13 illustrates a tonsil slide with an irradiated germinal center (no staining) next to another germinal center that was not irradiated (positive staining). The UV for LCM is 355 nm.

[0268] Example 5 Fluorescent detection of PNA / antibody conjugates using SA-FITC In addition to chromogenic detection, the biotin in the conjugate can also be fluorescently detected using SA-fluorophores. Figures 6A and 6B show fluorescent images of tonsil slides stained for Ki67 using a primary antibody followed by a PNA-conjugated secondary antibody and detected with SA-FITC. SA-FITC binds to the biotin on the PNA sequence (see Figure 6C). The fluorescent signal is consistent with the localization of the Ki67 marker. This experiment demonstrated the versatility of the detection scheme offered by PNA-conjugated antibodies.

[0269] Example 6 Quantitative measurement of PNA Without wishing to be bound by any particular theory, the NanoString nCounter platform has been reported to have the potential to detect up to 800 different DNA sequences, and the use of antibody-PNA conjugates has been shown to enable extremely large multiplexing capabilities for IHC. The inventors hypothesize that PNAs can be detected in a similar manner to DNA, since the detection scheme is based on hybridization of a reporter strand to a target oligo (which could potentially be DNA or PNA). Because the presence of biotin on the 3' end of PNAs eliminates the need for a capture strand, PNA oligomers could be significantly shorter than the standard DNA targets (typically about 100 bases long) typically used in the NanoString nCounter platform. Furthermore, the higher binding affinity of PNAs to DNA compared to DNA to DNA is thought to provide sufficient stability for the relatively short PNA / DNA reporter duplex.

[0270] As a proof of concept, we demonstrated that PNA-SA-FITC can be removed by incubating the fluorescently stained slides described in Example 8 herein with TCEP (20 mM). The released PNA-SA-FITC was measured based on fluorescence intensity. The number of PNA-conjugated antibodies bound to the slides can then be estimated (Figures 7A and 7B).

[0271] Example 7 Detection of PNA using complementary fluorescent DNA: In previous experiments, PNAs were detected by staining against the biotin label. Complementary DNA or PNA sequences carrying different labels (e.g., chromogenic, fluorogenic) can be used as alternative staining techniques. In this experiment, tonsil slides were incubated with rabbit anti-Ki67 primary antibody followed by PNA-conjugated GAR. The slides were then incubated with DNA sequences complementary to the fluorescently labeled PNAs (e.g., FITC or rhodamine). Figures 8A, 8B, 8C, and 8D show that the slides were successfully stained by DNA hybridization. Fluorescent staining was consistent with marker localization, and no background signal was observed. The DNA sequences were added as a manual titration at a concentration of approximately 185 nM and in a volume of approximately 100 μL.

[0272] DNA sequence: 1: 5'-CTGAAGATGGTTGAC / rhodamine-3' (SEQ ID NO: 16) 2: 5'-FAM / CTGAAGATGGTTGAC-3' (SEQ ID NO: 17)

[0273] This experiment demonstrates that antibody-conjugated PNA tags are active and accessible through hybridization with complementary DNA on tissue. Two DNA sequences were complementary to the same PNA, each carrying a unique fluorophore. The two fluorophores were placed at different ends of the DNA sequence (FITC at the 5' end and rhodamine at the 3' end). As a result, after hybridization with the PNA, FITC was located at the end farthest from the antibody, while rhodamine was located closer to the antibody. Positive staining was observed in both cases, demonstrating the ability to detect PNAs using hybridization to complementary DNA sequences.

[0274] In another experiment, tonsillar tissue was stained with anti-CD3 conjugated to 10-base PNA sequence 1 and anti-CD8 conjugated to 10-base PNA sequence 2 (PNA sequence 1 differs from PNA sequence 2). The two PNA-conjugated antibodies were added simultaneously as a cocktail. Two 10-base DNA sequences complementary to the two PNA sequences were then added as a cocktail, such that DNA sequence 1 was complementary to PNA sequence 1 and had an Alexa488 fluorophore at its end, and DNA sequence 2 was complementary to PNA sequence 2 and had an Alexa647 fluorophore at its end. Fluorescence images show that when we looked under the green channel, all CD3-positive cells were shown (Figure 27A), and when we looked under the red channel, all CD8-positive cells were shown (Figure 27B). The merged image shows the co-localization of the two markers (Figure 27C).

[0275] Example 8 Detection of PNA using complement-haptenized DNA In this experiment, complementary DNA was labeled with a hapten (DIG). Two tonsillar slides were incubated with rabbit anti-Ki67 and mouse anti-CD45, followed by PNA-conjugated GAR and PNA-conjugated GAM, respectively. DIG-labeled complementary DNA was incubated with both slides, followed by HRP conjugated to anti-DIG antibody and DAB deposition. DNA was added at a concentration of approximately 185 nM in a volume of approximately 100 μL at 37°C as a manual titration step.

[0276] Figures 9A, 9B, and 9C show that the slides were successfully stained and the staining pattern was consistent with the marker localization. No background signal was observed when the primary antibody was omitted.

[0277] DNA sequence: 5'-CTGAAGATGGTTGAC / DIG / -3' (SEQ ID NO: 18)

[0278] Figure 25 shows that a similar experiment was performed using a shorter PNA sequence (10 bases). Tonsil tissue was stained with anti-Ki67, then GAR conjugated to a short PNA (10 bases), and then incubated with a 10-base DNA sequence complementary to the 10-base PNA tag. The DNA sequence was stained with anti-DIG-HRP antibody and then labeled with DIG for visualization by DAB deposition.

[0279] Example 9 PNA Ab conjugation via click chemistry The steps for conjugating a PNA oligomer to an antibody are as follows:

[0280] (1) Antibody reduction to introduce sulfhydryl groups (thiols): Add 2.5 μL of 1 M DTT (dithiothreitol) to 100 μg of Ab and incubate for 30 minutes.

[0281] (2) Remove excess DTT using a Zeba desalting spin column (7 MWCO).

[0282] (3) Add DBCO-maleimide heterobifunctional linker (from click chemistry tool A108-25) at an Ab:linker ratio of 1:12 and incubate overnight.

[0283] (4) Clarify on a Zeba column, add azide-PNA at a 1:6 Ab:azide-PNA ratio, and incubate overnight.

[0284] (5) Clarify on a Zeba column.

[0285] Figure 19 shows tonsillar tissue incubated with anti-Ki67 primary antibody (rabbit mAb) and GAR-PNA (conjugated with click chemistry) and detected with SA-HRP. The image demonstrates that the PNA was successfully conjugated to the antibody via click chemistry.

[0286] Example 10 PNA Ab conjugation via maleimide chemistry The steps for conjugating a PNA oligomer to an antibody are as follows:

[0287] (1) Antibody reduction to introduce sulfhydryl groups (thiols): Add 2.5 μL of 1 M DTT (dithiothreitol) to 100 μg of Ab and incubate for 30 minutes;

[0288] (2) Remove excess DTT with a Zeba desalting spin column (7 MWCO);

[0289] (3) Add maleimide-PNA at a 1:6 Ab:maleimide DNA ratio and incubate overnight; and

[0290] (4) Clarify on a Zeba column.

[0291] Example 11 PNA quantification using Gyros Platform technology To demonstrate PNA quantification using Gyros technology, biotinylated PNA oligos (Bt-PNAs) were tested at concentrations ranging from approximately 0.0274 nM to approximately 20 nM (Table 1). For each concentration, a 1:2 ratio of Bt-PNAs to digoxigenin-labeled complementary single-stranded DNA (DNA-DIG) was first hybridized at room temperature and then analyzed by Gyros in duplicate. Detection was achieved using Ms-anti-DIG followed by Alexa Fluor 647-GAM. A four-point curve (see Table 1) was fitted using the Gyros program, yielding an R of >0.998. 2 In particular, the signal-to-background ratio (S / B) of the lowest PNA concentration was approximately 10, suggesting a very low background signal due to PNA or ssDNA. TIFF0007727385000018.tif253170

[0292] To further demonstrate that PNA cleaved from antibody conjugates can be quantified and that antibodies can be further stained after PNA cleavage, the experiment shown in Figure 33 was performed. Three antibodies (Ki67, CD8, and PD-L1) were conjugated to Bt-PNA-1 and used to detect the respective markers in normal tonsil tissue sections. Most tissue processing steps were performed using a Ventana BenchMark XT autostainer. After standard antigen retrieval, PNA-labeled primary antibodies were applied to the tissue and incubated at approximately 37°C for approximately 16 minutes. The slides were then removed from the tissue staining solution and rinsed with reaction buffer and water. Approximately 100 μL of 20 mM TCEP solution was added to each slide and incubated in a humidity box for approximately 20 minutes. Eighty microliters of the solution containing the cleaved PNA was collected and further quantified by Gyros (e.g., using the techniques described herein). After PNA cleavage, the slides were returned to the autostainer, and the primary antibodies were further detected using a Ventana UltraView DAB Universal Detection Kit.

[0293] Quantification of the cleaved Bt-PNAs was achieved by hybridization with an excess of complementary single-stranded DNA-digoxigenin, following the same procedure as previously described. The results are summarized in Table 2. The results clearly suggest that PNAs derived from antibody conjugates for tissue staining can be successfully cleaved and further quantified using the Gyros technology. Table 2: Quantification of PNA cleaved from tissues TIFF0007727385000019.tif142170

[0294] After PNA cleavage, the antibodies on the exact same tissue sections could still be stained by standard IHC to visualize the PNA-encoded markers, as shown in Figures 34A and 34B. Specific staining for each marker was observed, suggesting that the cleavage conditions did not cause significant damage to the tissue or to the antibody binding to the markers. This result suggests a new method for combining quantification and visualization of the spatial distribution of biomarkers in FFPE tissues, a unique advantage of antibody-PNA conjugates.

[0295] Example 12 - Comparison of different PNA oligomers detected by Gyros technology The procedure described in Example 11 was used. The following PNA and DNA oligomers were compared: PNA1: Biotin-o-GTCAACCATCTTCAG-Lys(C6SH)-3' sPNA1: Biotin-o-CCATCTTCAG-Lys(SMCC) Gamma PNA (gPNA): Biotin-O-GT * -CAA * -CCA * -TCT * -TCA * -G-Lys(SMCC) Short gamma PNA (sgPNA): Biotin-O-CCA * -TCT * -TCA * -G-Lys(SMCC) DNA:Biotin-GTCAACCATCTTCAG Detection cDNA-DIG: 5'-DIG-CTGAAGATGG-3' Final concentrations of PNA tested: 250 pM, 50 pM, 10 pM and 2 pM Final concentration of detection cDNA-DIG: 25 nM

[0296] PNA or DNA oligomers were incubated with cDNA-DIG at room temperature before analysis on the Gyros. The results of the analysis are shown in Figures 35A and 35B.

[0297] As shown in Figure 35A, all PNA oligomers could be detected at the lowest concentration (2 pM), but DNA oligomers could not be detected at the same concentration (low S / B ratio). At all concentrations, the response from PNA oligomers was much higher than that from DNA oligomers. This demonstrated the excellent stability of PNA-DNA hybrids. The difference in response between different PNAs was not significant.

[0298] Figure 35B shows that the signal-to-background (S / B) ratio of the PNA oligomers was much higher than that of the corresponding DNA oligomers, especially at lower concentrations (bottom panel). Overall, the results suggested that PNA oligomers (even 10-mer sequences) could be quantified down to low pM concentrations using Gyros. On the other hand, DNA oligomers (15-mers) with the same sequences could not be quantified down to such low pM concentrations. Furthermore, gamma PNA oligomers yielded higher S / B ratios compared to the corresponding PNA oligomers.

[0299] Example 13A In this example, tonsillar tissue was stained with Ki67 (rabbit Ab) followed by a goat anti-rabbit (GAR) antibody conjugated to a PNA sequence (CCATCTTCAG). A DNA sequence complementary to the PNA oligomer and bearing a DIG reporter moiety at its end was incubated with the GAR-PNA on the slide. After washing, an anti-DIG:HRP Ab was added. DAB was then deposited to produce a brown signal. The presence of a signal indicates successful hybridization of the DNA to the PNA, as the signal requires the presence of DIG on the DNA. The absence of a signal (as shown in Figure 36A) indicated that the DNA did not hybridize under these experimental conditions (200 nM DNA, 37°C, 30 min). It is believed that the affinity of DNA PNAs is not strong enough to ensure hybridization with such short PNA sequences under these experimental conditions.

[0300] Example 13B In this experiment, the PNA from Example 13A was replaced with a gamma PNA (15 bases, but only 10 bases are complementary to the same DNA sequence). The same experimental conditions as in Example 13A were applied. Compared with Example 12, the presence of a signal indicates that the gamma PNA / DNA duplex is even more stable than the PNA / DNA of the same hybridization sequence and under the same conditions (see Figure 36B). In fact, the presence of a signal indicated that the gamma DNA hybridized to the gamma PNA, producing a brown signal. Because the experimental conditions were the same as in Example 13A, this experiment showed that the affinity of the gamma PNA DNA was higher than that of the PNA DNA, which indicates the superiority of the gamma PNA compared to the short PNA, as in Example 13A.

[0301] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature referenced herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts from the various patents, applications, and publications to provide further embodiments.

[0302] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is thus understood that numerous modifications can be made to the exemplary embodiments and other arrangements can be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.

Claims

1. A composition for detecting a target in a sample, comprising a PNA conjugate having the structure of formula (IIB): [In the formula, The "specific binding entity" is selected from the group consisting of an antibody, an antibody fragment, a drug / antibody conjugate, and a nucleic acid; The "linker" has the structure shown in formula (IVA): [In the formula, d and e are each independently an integer ranging from 2 to 20; Q is a bond, O, or S; R a and R b are independently H, a C 1 -C 4 alkyl group, F, Cl, or N(R c )(R d ); R c and R d are independently CH 3 or H; A and B are independently branched or unbranched, straight chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having from 1 to 12 carbon atoms, and optionally having one or more O, N, or S heteroatoms; at least one of A or B comprises a cleavable moiety; A "PNA" is a PNA having at least one PNA nucleotide containing a substituent at the gamma carbon position; X is selected from the group consisting of biotin, an enzyme, a chromogen, a fluorophore, a hapten, and a mass spectrometry tag; Y is a branched or unbranched, straight chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms; m is 0; z is 0; and n is an integer ranging from 1 to 12.

2. The composition of claim 1 , wherein the "specific binder" is an antibody.

3. 3. The composition of claim 1 or 2, wherein the substituent of at least one substituted PNA nucleotide is selected from the group consisting of a lysine residue, a peptide having less than 20 amino acids, a polymer, and a miniPEG, and optionally, the substituent of at least one substituted PNA nucleotide further comprises at least one reporter moiety.

4. 4. The composition of claim 1, wherein m is 0, z is 0, and n is an integer greater than 1 and less than or equal to 12.

5. The composition of claim 4, wherein n is an integer ranging from 2 to 6.

6. The composition of claim 1 , wherein the "linker" comprises at least one hydrophilic group.

7. 7. The composition of claim 1, wherein d and e are integers ranging from 2 to 6.

8. The composition of claim 1 , wherein the cleavable moiety is a photocleavable group or a chemically cleavable group.

9. 1. A method for detecting a target in a sample in vitro, comprising: contacting the sample with a first PNA conjugate, the first PNA conjugate being the PNA conjugate of claim 1; contacting the sample with a first detection reagent to facilitate detection of the PNA conjugate; A method comprising:

10. 10. The method of claim 9, wherein the "specific binder" is a primary antibody, the primary antibody being specific for a first target, or the "specific binder" is a secondary antibody, the method further comprising contacting the sample with a primary antibody specific for the first target prior to contacting the sample with the first PNA conjugate, the first PNA conjugate being specific for the first primary antibody.

11. 11. The method of claim 9 or 10, wherein the first detection reagent is an anti-label antibody specific for the label of the PNA conjugate, optionally wherein the label is a hapten and the anti-label antibody is an anti-hapten antibody.

12. 12. The method of any one of claims 9 to 11, wherein the detection reagent comprises a PNA or DNA complementary to the PNA or gamma PNA of the first PNA conjugate, and the complementary PNA or DNA is conjugated to a reporter moiety.

13. 13. The method of claim 12, wherein the reporter moiety is a fluorophore or the reporter moiety is a hapten, and the method further comprises contacting the sample with an anti-hapten antibody specific for the complementary PNA or DNA hapten.

14. 10. The method of claim 9, wherein the method further comprises contacting the sample with a reagent to cleave the cleavable portion of the "linker" and quantifying the amount of cleaved "PNA."

15. 15. The method of claim 14, wherein quantification of the amount of PNA is performed using NanoString nCounter technology, Gyros technology, or mass spectrometry.

16. 16. The method of claim 14 or 15, wherein the cleavable moiety is selected from the group consisting of a photocleavable group, a chemically cleavable group, or an enzymatically cleavable group.

17. 17. The method of any one of claims 14 to 16, further comprising visualizing the specific binders in which the PNA has been cleaved.

18. A composition for detecting a target in a sample, comprising a conjugate of formula (IB): [In the formula, The "specific binding entity" is selected from the group consisting of an antibody, an antibody fragment, a drug / antibody conjugate, and a nucleic acid; The "linker" has the structure shown in formula (IVA): [In the formula, d and e are each independently an integer ranging from 2 to 20; Q is a bond, O, or S; R a and R b are independently H, a C 1 -C 4 alkyl group, F, Cl, or N(R c )(R d ); R c and R d are independently CH 3 or H; A and B are independently branched or unbranched, straight chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having from 1 to 12 carbon atoms, and optionally having one or more O, N, or S heteroatoms; at least one of A or B comprises a cleavable moiety; Z is a PNA having at least one PNA nucleotide containing a substituent at the gamma carbon position; X is selected from the group consisting of biotin, an enzyme, a chromogen, a fluorophore, a hapten, and a mass spectrometry tag; Y is a branched or unbranched, straight chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 1 to 12 carbon atoms and optionally having one or more O, N, or S heteroatoms; m is 0; z is 0; and n is an integer ranging from 1 to 12.

19. The composition of claim 18 , wherein the “specific binder” is a primary antibody.

20. 20. The composition of claim 18, wherein a substituent of at least one substituted PNA nucleotide is selected from the group consisting of a lysine residue, a peptide having less than 20 amino acids, a polymer, and a miniPEG, and optionally, at least one reporter moiety is linked to at least one substituted PNA nucleotide.

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