Ex vivo detection of protease activity in hepatocellular carcinoma

Abstract

The present application provides compositions and methods for determining hepatocellular carcinoma (HCC) in a subject, the methods including contacting a body fluid with a molecule that includes a reporter, the reporter being cleaved by an agent in the body fluid, the subject having or suspected of having cirrhosis of the liver.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 325,249, filed March 30, 2022, and U.S. Provisional Patent Application No. 63 / 331,515, filed April 15, 2022, both of which are incorporated by reference in their entireties. Summary of the Invention

[0002] Provided herein is a method comprising contacting a body fluid sample from a subject with a synthetic molecule, wherein the contacting occurs ex vivo, and the subject has or is suspected of having cirrhosis of the subject's liver, and the synthetic molecule comprises a cleavable linker, a reporter, and a carrier, and the synthetic molecule reacts with an enzyme from the body fluid to cause the reporter to form a detectable signal; and detecting the detectable signal, wherein the detection indicates whether the subject has hepatocellular carcinoma (HCC), and wherein the detection comprises a detection sensitivity of at least 0.5.

[0003] In some embodiments, the detection sensitivity is at least 0.8. In some embodiments, the detection sensitivity is calculated by dividing the number of samples identified as having HCC and being positive by the total number of samples, where the total number of samples includes the sum of the samples identified as having HCC and being positive, and the samples identified as having HCC and being negative.

[0004] In some embodiments, HCC comprises the stage of HCC. In some embodiments, an enzyme from the body fluid cleaves the cleavable linker, thereby releasing the reporter from the synthetic molecule, and the released reporter forms a detectable signal. In some embodiments, the cleavable linker comprises a peptide.

[0005] In some embodiments, the synthetic molecule comprises a panel of peptides. In some embodiments, the panel of peptides comprises at least 10 different peptides. In some embodiments, the enzyme comprises a protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP), dipeptidyl-peptidase 4 (DPP4), alanyl aminopeptidase (ANPEP), aminopeptidase (ERAP1, ARTS-1), kallikrein-related peptidase 14 (KLK14), cathepsin, or ADAM17.

[0006] In some embodiments, the reporter comprises a fluorescent label. In some embodiments, the fluorescent label comprises 5-carboxyfluorescein (5-FAM), 7-amino-4-carbamoylmethylcoumarin (ACC), 7-amino-4-methylcoumarin (AMC), 2-aminobenzoyl (ABZ), Cy7, Cy5, Cy3, or (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid) (EDANS). In some embodiments, the synthetic molecule further comprises a fluorescence quencher. In some embodiments, the fluorescent quencher comprises BHQ0, BHQ1, BHQ2, BHQ3, BBQ650, ATTO 540Q, ATTO 580Q, ATTO 612Q, CPQ2, QSY-21, QSY-35, QSY-7, QSY-9, DABCYL (4-([4'-dimethylamino)phenyl]azo)benzoyl), Dnp (2,4-dinitrophenyl), or Eclipse.

[0007] In some embodiments, the bodily fluid sample comprises blood, plasma, bone marrow fluid, lymphatic fluid, bile, amniotic fluid, mucosal fluid, saliva, urine, cerebrospinal fluid, spinal fluid, synovial fluid, semen, ductal aspirate, feces, stool, vaginal discharge, tears, tissue lysate, or patient-derived cell line supernatant. In some embodiments, the bodily fluid sample comprises rinse, conditioning medium or buffer, swab viral transport medium, saline, culture medium, or cell culture supernatant. In some embodiments, the rinse comprises mouthwash rinse, bronchoalveolar rinse, lavage fluid, hairwash rinse, nasal spray discharge, swab of any body surface, orifice, organ structure, or solid tumor biopsy applied to saline or any medium, or any derivative thereof.

[0008] In some embodiments, the bodily fluid sample comprises an alpha fetoprotein (AFP) level of less than about 10 ng / ml. In some embodiments, detecting further comprises detecting the AFP level of the bodily fluid sample. In some embodiments, detecting comprises detecting the rate of formation or amount of a detectable signal. In some embodiments, the subject comprises a mammal. In some embodiments, the subject comprises a human.

[0009] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (herein referred to as "Figures"), in which: [Brief description of the drawings]

[0011] [Figure 1]Shown are multiple probes according to the present application. Each probe 101 includes a reporter 103, shown as a star in Figure 1. The reporter 103 is attached to a cleavable linker 105 that is a cleavable substrate for a drug 107. [Diagram 2] 1 shows cleavage of reporters in a plurality of probes. As shown, cleavage of the cleavable linker 105 by an agent 107 results in the reporter 103 being cleaved from the probe 101. Upon cleavage, the cleaved reporter 203 can be detected and / or distinguished from the uncleaved reporter 103. The presence and detection of the cleaved reporter 203 indicates that the agent 107 is present and active in the sample. Additionally, the absence of agent activity can be used to detect associated decreased activity. The activity of the agent can be quantified, for example, based on the rate at which the cleavage reaction occurs or the amount of cleaved reporter in the sample, or by such means as the ratio of the rate to an appropriate control or the ratio of the cleaved reporter to an appropriate control. [Diagram 3] A method 301 for assessing a biological condition in a subject using a probe 101 is shown. [Figure 4] 4 shows a selection of probes for use in a composition to analyze the activity of a drug to analyze one or more specific biological or disease states. The activity of one or more drugs can be associated with a biological or disease state. This can include the progression of a particular condition or state over time. Thus, probes that can be cleaved by a drug of interest to assess a biological or disease state in a subject are selected from a library for inclusion in a condition-specific panel 403. The selected probes 405 of the condition-specific panel are differentially labeled 305 so that the activity of a given protease can be measured. Different probes 101, including those included in the library 401, can include features that impart properties to the fragments to ensure accurate multiplex detection of drug activity. Such properties can include, for example, improved cleavage, detection, solubility, stability, reproducibility, robustness, and / or expanded compatibility with different types of reporters. [Diagram 5] We show that protease activity significantly distinguishes hepatocellular carcinoma (HCC) samples versus healthy samples in two types of samples: virus-induced HCC (FIG. 5) and Western (metabolically-driven) HCC (FIG. 6). [Figure 6] We show that protease activity significantly distinguishes hepatocellular carcinoma (HCC) samples versus healthy samples in two types of samples: virus-induced HCC (FIG. 5) and Western (metabolically-driven) HCC (FIG. 6). [Figure 7] 1 shows that protease activity significantly distinguishes HCC versus cirrhosis samples. [Figure 8] We show that the HCC signal generalizes across sample cohorts without classifier retraining, increasing confidence in the results. [Figure 9] Figure 2 shows that protease activity varies with cancer stage in HCC, both in virus-induced HCC (A) and lifestyle-induced HCC (B). [Figure 10] 1 shows the results of an AFP protease biosensor panel assay. A shows the results of a cross-validated classifier in low AFP patients (AFP<10 ng / ml). 0B shows the results of a combined classifier using a protease panel + AFP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Methods are provided herein that include contacting a body fluid sample from a subject with a synthetic molecule (also referred to herein as a "molecule" or "probe") ex vivo, in vitro, or in vivo. In some embodiments, the molecule includes a cleavable linker and a reporter. The cleavable linker is cleaved by an agent from the body fluid to release the reporter from the molecule. In some embodiments, the method further includes detecting a formation rate or amount of the released reporter. In some embodiments, the formation rate or amount of the released reporter is significantly different from that of a healthy subject. In some embodiments, the body fluid includes plasma. In some embodiments, the method further includes determining a disease or condition of the subject based on the detection. In some embodiments, the disease or condition includes hepatocellular carcinoma (HCC). In some embodiments, the subject has or is suspected of having cirrhosis of the liver. In some embodiments, the method includes distinguishing between subjects with HCC and subjects with only cirrhosis of the liver.

[0013] In one aspect, the bodily fluid sample is contacted with the second cleavable linker and the second reporter by the second molecule. In some embodiments, the second cleavable linker is cleaved by the second agent from the bodily fluid, releasing the second reporter from the second molecule. In some embodiments, the method further comprises detecting the rate or amount of formation of the released second reporter. In some embodiments, the method further comprises determining the disease or condition of the subject based on the detection of the first released reporter and the detection of the second released reporter. In some embodiments, the methods described herein can be used in a multiplexed format, such that the activity of multiple selected agents can be confirmed using a single bodily fluid sample. This allows diagnostic panels for specific pathologies and conditions to be created, leveraging the activity of multiple agents to provide a more complete and accurate assessment of a particular condition. These panels can be used to correlate the activity of multiple agents with a particular condition or disease state. These signatures can be stored, for example, in a database and used to assess the condition or disease state of subsequent individuals assessed by a particular protease activity panel. In some embodiments, classification tools are used in the analysis to distinguish between healthy and diseased patients or between distinct stages of disease. Classification tools can be supervised machine learning classification algorithms, including but not limited to logistic regression, naive Bayes, support vector machines, random forests, gradient boosting, or neural networks. Furthermore, when the modeled variables are continuous in nature (e.g., tumor volume), continuous regression approaches such as ridge regression, kernel ridge regression, or support vector regression can be used. These algorithms operate on a multidimensional feature space defined by the measurements of multiple probes (or mathematical functions of those measurements, such as probe ratios) to learn the relationship between the probe measurements and the disease state. Finally, the probe measurements can be combined with clinical variables such as age, sex, or the patient's comorbidity status.One can then either directly incorporate the clinical features in the classifier, or alternatively, learn a secondary classifier that combines the probe-only predictions with the clinical features to produce results that are calibrated to those variables.

[0014] In some embodiments, the disease or condition comprises liver disease, cancer, organ transplant rejection, infectious disease, allergic disease, autoimmunity, and chronic inflammation. In some embodiments, the liver disease comprises a particular fibrosis stage of non-alcoholic steatohepatitis (NASH) or a particular non-alcoholic fatty liver disease activity score (NAS).

[0015] In another aspect, the methods described herein include ex vivo multiplex detection of enzyme activity for diagnosing and monitoring pathology and treatment in a subject, which enzyme activity can be used to diagnose and monitor diseases and conditions in internal organs of a subject.

[0016] Detection probes / molecules The determination of the disease or condition is based on the rate of formation or amount of released reporter detected in the sample. A probe / molecule is introduced into the bodily fluid sample. The probe / molecule comprises a cleavable linker and a reporter, and an agent from the bodily fluid cleaves the cleavable linker and releases the cleaved reporter. The probe / molecule may have any structure capable of performing this function. In some embodiments, the reporter may be covalently attached to the cleavable linker. In some embodiments, the reporter comprises a fluorescent label, a mass tag, a chromophore, an electrochemically active molecule, a biolayer interferometry or surface plasmon resonance detectable molecule, a precipitating substance, a mass spectrometry and liquid chromatography substrate (including size exclusion, reversed phase, isoelectric point, etc.), a magnetically active molecule, a gel-forming and / or viscosity-changing molecule, an immunologically detectable molecule, a cell-based amplification detectable molecule, a nucleic acid barcode, or any combination thereof.

[0017] In some embodiments, the reporter comprises a fluorescent label and the molecule also comprises a quencher. In some embodiments, the quencher is covalently attached to the cleavable linker. In some embodiments, an internally quenched fluorophore is attached to the cleavable linker. In some embodiments, the molecule further comprises a self-immolative spacer. In some other embodiments, the molecule further comprises a carrier.

[0018] Cleavable Linkers In some aspects, the probe / molecule described herein comprises a cleavable linker. The cleavable linker described herein can be any structure that can be cleaved by an agent. In some embodiments, the cleavable linker comprises a peptide, a carbohydrate, a nucleic acid, a lipid, an ester, a glycoside, a phospholipid, a phosphodiester, a nucleophile / base sensitive linker, a reduction sensitive linker, an electrophile / acid sensitive linker, a metal cleavable linker, an oxidation sensitive linker, a self-immolative linker (three-component probe=enzyme substrate+linker+reporter), or a combination thereof. In some embodiments, the reporter can be in an inactive form and becomes detectable under disease activity. Geoffray Leriche, Louise Chisholm, Alain Wagner, Cleavable linkers in chemical biology, Bioorganic&Medicinal Chemistry, Volume 20, Issue 2, 2012, Pages 571-582, ISSN 0968-0896, https: / / doi.org / 10.1016 / j.bmc.2011.07.048.

[0019] Crosslinkers aim to form covalent bonds between two spatially adjacent residues in one or two polymer chains. To identify protein binding partners, crosslinkers need to be able to detect and stabilize transient interactions. Crosslinkers frequently form covalent bonds between lysine or cysteine ​​residues in proteins. Alternatively, crosslinkers can be photoreactive. Crosslinking cleavable linkers can be used to differentiate between and within protein-protein interactions in receptors, signaling cascades, and structures of multi-protein complexes.

[0020] In some embodiments, the cleavable linker comprises a peptide. The core structure of the peptide linker may comprise either a dipeptide or a tetrapeptide that is recognized and cleaved by lysosomal enzymes. Proteases (also called peptidases) catalyze the breakdown of peptide bonds by hydrolysis and are restricted to specific sequences of amino acids that can be recognized by the protease. Commonly used proteases include pepsin, trypsin, or chymotrypsin. Because proteases play an important role in many diseases, peptide linkers are widely used in drug release systems or diagnostic tools. In some embodiments, the peptide linker comprises a short peptide sequence. In some embodiments, the peptide linker comprises at least one non-naturally (also referred to as "unnaturally") occurring amino acid.

[0021] In some embodiments, the peptide linker comprises less than about 20 amino acids in length. In some embodiments, the peptide linker comprises between 10 and 100 amino acids in length. In some embodiments, the peptide linker comprises an amino acid length of 1-5, 1-10, 1-20, 1-30, 1-50, 1-70, 1-90, 1-100, 5-10, 5-20, 5-30, 5-50, 5-70, 5-90, 5-100, 10-20, 10-30, 10-50, 10-70, 10-90, 10-100, 20-30, 20-50, 20-70, 20-90, 20-100, 30-50, 30-70, 30-90, 30-100, 50-70, 50-90, 50-100, 70-90, 70-100, or 90-100. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

Table 1-13

Table 1-14

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Table 1-16

Table 1-17

Table 1-18

Table 1-19

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Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

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Table 1-29

[0022] In some embodiments, the peptide linker described herein for an endoprotease has the design: m AY n or AX n Each of the peptide linkers includes a single amino acid, A and B are an amino acid pair recognized by a specific endoprotease, X and Y include any amino acid that may or may not be labeled with a reporter, and m and n include any integer, including zero. This design is for illustrative purposes only and should not be construed as the only possible design for a peptide linker.

[0023] In some embodiments, the peptide linkers described herein for the exoproteases have the design: m AY n where A comprises an amino acid pair recognized by a specific exoprotease, X and Y comprise any amino acid that may or may not be labeled with a reporter, and n and m comprise any integer, including zero. This design is for illustrative purposes only and should not be construed as the only possible design for a peptide linker. [Table 2-1] [Table 2-2]

[0024] In some embodiments, the cleavable linker comprises a carbohydrate. Tung et al. reported a conjugate of β-galactosides and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) with far-red fluorescent properties after cleavage by β-galactosidase. Tung CH, Zeng Q, Shah K, Kim DE, Schellingerhout D, Weissleder R. In vivo imaging of beta-galactosidase activity using far red fluorescent switch. Cancer Res. 2004 Mar 1; 64(5): 1579-83. Ho et al. reported combining a β-galactosidase substrate with p-benzyloxycarbonyl as a self-immolative linker. β-D-galactopyranoside, a substrate for β-galactosidase, was conjugated to the optical probe via a para-substituted benzyloxycarbonyl group (serving as the first self-immolative linker) and a glycine residue (serving as the quencher and second self-immolative linker). Enzymatic cleavage of β-D-galactopyranoside triggered a series of spontaneous reactions resulting in the release of the optically active probe. Ho, N.-H., Weissleder, R. and Tung, C.-H. (2007), A Self-Immolative Reporter For β-Galactosidase Sensing. ChemBioChem, 8:560-566. Several carbohydrate linkers are commercially available.

[0025] In some embodiments, the cleavable linker comprises a nucleic acid. The effect of the DNA linker on the behavior of the conjugate is to reduce the toxicity of the free drug by reducing its cellular penetration, which is positive in the case of early deconjugation in the bloodstream, and to increase off-target toxicity to low antigen expressing cells, possibly due to non-specific interactions of the nucleic acid-based linker with the cell surface. For example, in an antibody-drug conjugate, the antibody and the drug can be non-covalently linked using a complementary DNA linker. Dovgan, I., Ehkirch, A., Lehot, V. et al. On the use of DNA as a linker in antibody-drug conjugates: synthesis, stability and in vitro potency. Sci Rep 10, 7691 (2020). Dovgan et al. disclosed trastuzumab conjugated to monomethyl auristatin E (MMAE) by a 37-mer oligonucleotide.

[0026] In some embodiments, the cleavable linker comprises a lipid. In some embodiments, the cleavable linker comprises a phospholipid. Insertion of a phospholipid group between two fluorescent dyes or a dye / quencher pair allows for detection of phospholipase cleavage activity. In some embodiments, the cleavable linker comprises a phosphodiester. Insertion of a phosphodiester group between two fluorescent dyes or a dye / quencher pair allows for detection of phosphodiesterase cleavage activity. In some embodiments, the lipid is directly attached to the fluorophore: when the covalent bond between the lipid and the fluorophore is cleaved, an increase in fluorescence activity allows for detection of the presence of the enzyme.

[0027] In some embodiments, the cleavable linker comprises an ester. Ester groups are often cleaved by saponification. The reactivity of esters to cleavage can be enhanced by the use of electron-withdrawing groups or stabilized by the use of self-immolative spacers to preclude spontaneous hydrolysis. In chemical biology, ester-based cleavable compounds were used early on for protein purification and in structural biology. FRET-based probes were designed to image esterase activity.

[0028] In some embodiments, the cleavable linker comprises a glycoside. For example, cellulase enzymes break down cellulose into glucose and are often composed of a glycosylated linker that connects a glycoside hydrolase (GH) to a carbohydrate-binding module (CBM).

[0029] In some embodiments, the cleavable linker comprises a nucleophile / base sensitive linker, which may include, but is not limited to, a halogen nucleophile, an oxygen nucleophile, a safety catch linker, a thiol nucleophile, a nitrogen nucleophile, and a phenacyl ester derivative.

[0030] In some embodiments, the cleavable linker may be susceptible to activity from all enzyme families, including, but not limited to, oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.

[0031] Fluoride decomposable linkers are widely used in organic chemistry as silicon-based protecting groups for alcohols. The high thermodynamic affinity of fluorine for silicon allows for their removal in orthogonal and mild conditions using a fluoride source. In this reaction, fluoride ions react with silicon as a nucleophilic species, and the cleavage conditions depend on the steric hindrance of the alkyl groups of silicon. Fluoride ions can also cause bond cleavage due to their basic properties.

[0032] Oxygen nucleophiles include sulfone and ester linkers, but safety-catch linkers allow for greater control over the timing of bond cleavage because the linker remains stable until activated for cleavage by chemical modification.

[0033] In secondary amine synthesis or solid-phase synthesis, nitrobenzenesulfonamides are known to be cleaved with thiol nucleophiles such as b-mercaptoethanol. Cysteines can be modified with electron-deficient alkynes to form vinyl sulfide bonds.

[0034] Substitution reactions involving certain nitrogen species as nucleophiles can occur under mildly cleavable conditions. These reactions can be categorized into two groups: aminolytic cleavage or exchange reactions. For aminolytic cleavage, examples include the cleavage of malondialdehyde (MDA) indole derivatives with either pyrrolidine or hydrazine, and the cleavage of ester linkers with hydroxylamine or hydrazine. Acylhydrazones 44 and hydrazones 45, 156 can be used as linkers cleavable by imino transfer in mildly acidic media. Amine catalysts (e.g., aniline, p-anisidine, or hydroxylamine) accelerate the hydrolysis, allowing for an efficient transition between stable and dynamic states required for cleavage and exchange.

[0035] In some embodiments, the cleavable linker comprises a reduction-sensitive linker. Reduction-sensitive bonds have been used for a long time in chemical biology and are a commonly used class of cleavable linkers. Examples of cleavable linkers sensitive to reducing conditions include nitroreductase, disulfide bridges, and azo compounds. Karan et al. reported a fluorescent probe for detecting nitroreductase. Sanu Karan, Mi Young Cho, Hyunseung Lee, Hwunjae Lee, Hye Sun Park, Mahesh Sundararajan, Jonathan L.Sessler, and Kwan Soo Hong. Near-Infrared Fluorescent Probe Activated by Nitroreductase for In Vitro and In Vivo Hypoxic Tumor Detection. Journal of Medicinal Chemistry 2021 64(6), 2971-2981. In naturally occurring proteins, disulfide bridges generally play a role in maintaining protein structure. They are known to be efficiently and rapidly cleaved by mild reducing agents such as dithiothreitol (DTT), b-mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP). In chemical biology, disulfide bridges are used in a wide range of applications including functional and structural proteomics, drug delivery, tumor imaging, DNA and protein-DNA complex purification. Disulfide-based cleavable linkers are commonly used due to their simple synthesis and rapid cleavage. Azo linkers are highly attractive to chemical biologists because they can undergo cleavage after treatment with sodium dithionite, a mild and potentially bioorthogonal reducing agent. Azo compounds are reduced to two aniline moieties via an electrochemical reduction mechanism, which allows the use of reducing agents commonly used in many biological protocols such as TCEP, DTT, etc. In chemical biology, azo compounds have been used to crosslink proteins for over a decade, and more recently for protein affinity purification.

[0036] In some embodiments, the cleavable linker comprises an electrophile / acid-sensitive linker. Acid-sensitive linkers can be combined with other types of linkers. For example, the first β-galactosidase cleavage of β-D-galactopyranoside causes the self-immolation of the benzyloxycarbonyl group, resulting in the release of the optically active probe. Ho, N.-H., Weissleder, R. and Tung, C.-H. (2007), A Self-Immolative Reporter For β-Galactosidase Sensing. ChemBioChem, 8:560-566. Two different modes of electrophilic cleavage have been used in chemical biology: acid-sensitive linkers sensitive to proton sources and 2-(diphenylphosphino)benzoic acid alkyl derivatives sensitive to azide compounds. Proton-sensitive bonds are one of the most frequently used cleavable functions in organic chemistry, exemplified by the BOC group protecting amines, or the development of Merrifield resins used in solid-phase synthesis. In organic chemistry, acceptable cleavage conditions are very flexible with respect to acid reagents, solvents, temperature, and pH. In contrast, biocompatible acid-cleavable linkers must be responsive to slight changes in pH. Strong acidic conditions can result in denaturation of proteins and DNA. Biocompatible acid-cleavable linkers are selected for their instability near physiological pH, and are different from classical protecting groups, which are often cleaved with strong acids. Chemical reactions that can break or form bonds in water, such as the Staudinger ligation, can be used as the basis for cleavable linkers. This reaction proceeds by nucleophilic attack of a 2-(diphenylphosphino)benzoic acid alkyl derivative on the azide, forming an aza-ylide intermediate. An ester then captures the aza-ylide, which leads to the formation of an amide. In this process, the ester acts as the cleavable linker and the azide as the bioorthogonal chemical agent, ensuring chemoselective and bioorthogonal cleavage.

[0037] In some embodiments, the cleavable linker comprises a metal or metallic cleavable linker. Organometallic compounds are used to catalyze the modification of proteins containing unnatural amino acids, but their use as cleavage reagents in chemical biology has only been reported a few times. Allyl functions are commonly used protecting groups for alcohols in organic synthesis and are also used as cleavable linkers in DNA sequencing by synthesis. Metal cleavable linkers were also used in the design of peptide nucleic acids (PNAs), which were developed for enzyme-independent DNA / RNA hybridization methods.

[0038] In some embodiments, the cleavable linker comprises an oxidation-sensitive linker. Sodium periodate is undoubtedly the most frequently used biocompatible oxidizing agent due to its ability to cleave vicinal diols to form two aldehyde compounds. One example of this type of cleavable linker consists of a vicinal diol with a tartaric acid spacer and two functional groups at both ends. Selenium-based linkers also contain cleavable bonds that are sensitive to oxidizing agents, such as sodium periodate or N-chlorobenzenesulfonamide immobilized on polystyrene beads (iodobeads). The triggering agent oxidizes the labile bond to selenium oxide, which is then directly cleaved via intramolecular b-elimination or rearrangement.

[0039] Reporters and detection methods In some embodiments, the probe / molecule described herein comprises a reporter. The reporter described herein comprises or is comprised in any structure that may be detectable by any method, including but not limited to fluorescent detection, spectroscopic detection, immunological detection, or imaging detection. In some embodiments, the reporter comprises a fluorescent label, a mass tag, or a nucleic acid barcode.

[0040] In some embodiments, the reporter comprises a fluorescent label. Labels, tags, and probes, including small compounds such as fluorescers, can be used to label proteins and nucleic acids. Bioaffinity for other molecules (biotin, digoxigenin), enzymes (AP, HRP), or chemiluminescence (esters or acridines) can also be used. Genetically encoded markers such as the GFP family of fluorescent proteins have become the reporters of choice for gene expression studies and protein localization. In combination with subcellular tags, GFP can be used to label subcellular structures such as synapses, allowing new approaches to study developmental processes such as synapse formation. Other fluorescent labels include, but are not limited to, small organic dyes and lipophilic dyes. Fluorescent labels can function as active substrates themselves without the addition of a linker.

[0041] Some reporters are "internally quenched" and thus do not require a quencher; cleavage of the bond connecting the internally quenched fluorophore to the substrate linker yields the fluorescent molecule directly. Many described probes for proteases, esterases, peroxidases, and others function in this way.

[0042] In some embodiments, the reporter comprises a mass tag. Mass tag reagents are designed to allow identification and quantification of proteins in different samples using mass spectrometry (MS). Mass tagging reagents in a set typically have the same nominal mass (i.e., are isobaric) and chemical structure consisting of an amine-reactive NHS ester group, a spacer arm (mass normalization factor), and a mass reporter.

[0043] In some embodiments, the reporter comprises a nucleic acid barcode. For example, DNA barcoding is a system for species identification that focuses on the use of short, standardized genetic regions that act as "barcodes," in a manner similar to how the Universal Product Code is used by supermarket scanners to distinguish commercial products.

[0044] In some embodiments, the reporter can be detected using a ligand binding assay. Ligand binding assays often involve a detection step, such as ELISA, test strips or lateral flow assays, including fluorescent, colorimetric, bioluminescent, and chemiluminescent ELISAs, or bead-based fluorescent assays. In some embodiments, a paper-based ELISA test can be used to detect the cleaved reporter in a fluid sample. A paper-based ELISA can be inexpensively created, such as by reflowing wax deposited from a commercial solid ink printer to create an array of test spots on a single piece of paper. When the solid ink is heated to a liquid or semi-liquid state, the printed wax penetrates the paper and a hydrophobic barrier is formed. The spaces between the hydrophobic barriers can then be used as individual reaction wells. An ELISA assay can be performed by drying a detection antibody on the individual reaction wells to form a test spot on the paper, followed by a blocking and washing step. Fluid from a sample taken from a subject can then be added to the test spot. For example, a streptavidin alkaline phosphate (ALP) conjugate can then be added to the test spot as a detection antibody. The bound ALP can then be exposed to a colored reactant such as BCIP / NBT (5-bromo-4-chloro-3″-indolylphosphate p-toluidine salt / nitro-blue tetrazolium chloride), which causes a purple precipitate to form, indicating the presence of the reporter.

[0045] In some embodiments, the reporter can be detected using volatile organic compounds. Volatile organic compounds can be detected by analytical platforms such as gas chromatography instruments, breathalyzers, mass spectrometers, or the use of optical or acoustic sensors. Gas chromatography can be used to detect compounds that can be evaporated without decomposition (e.g., volatile organic compounds). Gas chromatography instruments contain a mobile phase (or moving phase), which is a carrier gas, e.g., an inert gas such as helium or a non-reactive gas such as nitrogen, and a stationary phase, which is a microscopic layer of liquid or polymer on an inert solid support inside a glass or metal tube called a column. The column is coated with the stationary phase, and the analyzed gaseous compounds interact with the walls of the column, causing them to elute at different times (i.e., have varying retention times in the column). Compounds can be differentiated by their retention times.

[0046] Mass spectrometry and enrichment / chromatography methods can be used to separate and differentiate / detect cleaved from intact reporters used in the present invention based on mass differences and / or the presence of labels. For example, enzymatic reactions can result in fragmentation of the parent molecule resulting in a mass shift of the starting substrate, which can be exploited in different chromatographic / enrichment methods such as size exclusion chromatography and affinity enrichment. In mass spectrometry, a sample is ionized, for example, by bombarding with electrons. The sample can be solid, liquid, or gas. By ionizing the sample, some of the molecules in the sample are broken down into charged fragments. These ions can then be separated according to their mass-to-charge ratio. This is often done by accelerating the ions and subjecting them to an electric or magnetic field, and ions with the same mass-to-charge ratio undergo the same amount of deflection. When deflected, the ions can be detected by a mechanism capable of detecting charged particles, for example, an electron multiplier. The detection results can be displayed as a spectrum of the relative abundance of the detected ions as a function of mass-to-charge ratio. Molecules in a sample can then be identified by correlating known masses, such as the mass of the entire molecule, to the identified masses, or by characteristic fragmentation patterns.

[0047] When the reporter comprises a nucleic acid, the reporter can be detected by various sequencing methods known in the art, such as conventional Sanger sequencing methods, or next-generation sequencing (NGS). NGS generally refers to non-Sanger-based high-throughput nucleic acid sequencing technology that can sequence a large number (i.e., thousands, millions, or billions) of nucleic acid strands in parallel. Examples of such NGS sequencing include platforms produced by Illumina (e.g., HiSeq, MiSeq, NextSeq, MiniSeq, and iSeq 100), Pacific Biosciences (e.g., Sequel and RSII), and Ion Torrent by ThermoFisher (e.g., Ion S5, Ion Proton, Ion PGM, and Ion Chef systems). It should be understood that any suitable NGS sequencing platform can be used for NGS to detect the nucleic acid of the detectable analyte described herein.

[0048] The analysis can be performed directly on the biological sample, or the detectable cleaved reporter can be first purified to some extent. For example, the purification step can include isolating the detectable analyte from other components in the biological sample. Purification can include methods such as affinity chromatography. The isolated or purified detectable analyte does not need to be 100% pure or substantially pure before analysis. Detecting the cleaved reporter can provide a qualitative assessment (e.g., whether the detectable cleaved reporter, and thus the given protease, is present or absent) or a quantitative assessment (e.g., the amount of detectable cleaved reporter present) to indicate the relative activity level of the given protease in the fluid sample. The quantitative value can be calculated by any means, such as determining the relative percent amount of each fraction present in the sample. Methods for performing these types of calculations are known in the art.

[0049] The cleaved reporter can be detected by any detection method that may be suitable for a particular reporter. In some embodiments, the detection method comprises fluorescence detection, spectroscopic detection, mass spectrometry, immunological detection, or imaging detection. In some embodiments, the detection method comprises fluorescence resonance energy transfer (FRET).

[0050] In some embodiments, the detection method includes spectroscopic detection. Spectroscopic methods of detection are very commonly used in ion chromatography (IC) and are second only to conductivity detection in their frequency of use. These methods can be broadly divided into the categories of molecular and atomic spectroscopy techniques. Molecular spectroscopy includes UV-visible spectrophotometry, refractometry, and photoluminescence techniques (fluorescence and phosphorescence). Atomic spectroscopy includes atomic emission spectroscopy (using various excitation sources) and atomic absorption spectroscopy. Many of the spectroscopic detection methods can operate in direct or indirect modes. The definitions of these terms are the same as those used to describe electrochemical detection modes. That is, when the solute ions have a greater value of the measured detection parameter than the eluent ions, it is a direct spectroscopic detection result. When the opposite is true, it is an indirect detection result.

[0051] In some embodiments, the detection method comprises mass spectrometry. Mass spectrometry (MS) is an analytical technique used to measure the mass-to-charge ratio of ions. The results are typically presented as a mass spectrum, a plot of intensity as a function of mass-to-charge ratio.

[0052] In some embodiments, the detection method involves fluorescence resonance energy transfer (FRET). FRET (fluorescence resonance energy transfer) is a distance-dependent dipole-dipole interaction that results in the transfer of energy from an initially excited donor molecule to an acceptor molecule without the emission of a photon. It allows the detection of molecular interactions in the nanometer range. FRET peptides are labeled with a donor molecule and an acceptor (quencher) molecule. In most cases, the donor and acceptor pair are two different dyes. If the acceptor is a non-fluorescent dye (quencher), the transferred energy from the fluorescent donor is converted into molecular vibrations. When FRET is terminated (by separating the donor and acceptor), an increase in donor fluorescence can be detected. When both donor and acceptor dyes are fluorescent, the transferred energy is emitted as light of a longer wavelength, allowing the change in the intensity ratio of donor and acceptor fluorescence to be measured. For efficient FRET quenching to occur, the fluorophore and quencher molecules must be in close proximity to each other (approximately 10-100 Å) and the absorption spectrum of the quencher must overlap with the emission spectrum of the fluorophore.

[0053] Precipitable fluorophores In some embodiments, the cleaved reporter comprises a precipitable fluorophore. In some embodiments, the precipitable fluorophore can be HPQ, Cl-HPQ, HTPQ, HTPQA, HBPQ, or HQPQ.

[0054] In some embodiments, the precipitating fluorophore comprises HPQ, also known as 2-(2"-hydroxyphenyl)-4(3H)-quinazolinone. HPQ is a small organic dye known for its classical emission mechanism via excited-state intramolecular proton transfer (ESIPT) and exhibits strong luminescence in the solid state but no luminescence in solution. HPQ was found to be strictly water insoluble and exhibits strong solid-state fluorescence similar to that of tetraphenylethylene. Furthermore, its intrinsic properties of insolubility and strong solid-state fluorescence can be countered and reversed by preventing the establishment of an internal hydrogen bond between the imine nitrogen and the phenolic hydroxyl group.

[0055] In some embodiments, the precipitating fluorophore comprises Cl-HPQ. Cl-HPQ is released when HPQF, a water-soluble non-fluorescent molecule, reacts with furin. Cl-HPQ begins to precipitate near the enzyme active site, and the precipitate emits bright solid-state fluorescence with over 60-fold fluorescence enhancement. Li et al. In Situ Imaging of Furin Activity with a Highly Stable Probe by Releasing of Precipitating Fluorochrome. Anal. Chem. 2018, 90, 19, 11680-11687.

[0056] In some embodiments, the precipitating fluorophore comprises HTPQ. HTPQ was found to be strictly water insoluble and exhibits strong fluorescence in the solid state with excitation and emission maxima of 410 nm and 550 nm, respectively. This makes it much more suitable for use in confocal microscopy. The large Stokes shift of HTPQ contributes additional highly desirable advantages: increased sensitivity, minimized background fluorescence, and enhanced bioimaging contrast. Liu et al.In Situ Localization of Enzyme activity in Live Cells by a Molecular Probe Releasing a Precipitating Fluorochrome.Angew Chem Int Ed Engl.2017 Sep 18;56(39):11788-11792.

[0057] In some embodiments, the precipitating fluorophore comprises HTPQA. HTPQA is another enzyme-responsive fluorogenic probe derived from HTPQ. When converted by ALP, the probe releases free HTPQ, which begins to precipitate after a very short delay, and the precipitate emits bright solid-state fluorescence with more than 100-fold fluorescence enhancement.

[0058] In some embodiments, the precipitating fluorophore comprises HBPQ, which is completely water insoluble and exhibits strong yellow solid-state emission when excited with a 405 nm laser. Liu et al. Precipitated Fluorophore-Based Molecular Probe for In Situ Imaging of Aminopeptidase N in Living Cells and Tumors. Anal. Chem. 2021, 93, 16, 6463-6471, Published: April 14, 2021.

[0059] In some embodiments, the precipitating fluorophore comprises HQPQ. HQPQ is a novel solid-state fluorophore that is water insoluble. Li et al. Precipitated Fluorophore-Based Probe for Accurate Detection of Mitochondrial Analytes. Anal. Chem. 2021, 93, 4, 2235-2243. Published: January 5, 2021.

[0060] Precipitating and non-precipitating fluorophores can be separated from the enzyme substrate by a self-immolative substrate to stabilize the initial probe and ensure that enzymatic cleavage is transduced via the sacrificial spacer into the formation of a precipitating fluorophore or a non-internally quenched soluble fluorophore.

[0061] Fluorescence quencher In some aspects, the probes / molecules described herein comprise a fluorescence quencher. The fluorescence quencher described herein can be any structure that can reduce the fluorescence intensity of a given substance. In some embodiments, the fluorescence quencher comprises BHQ0, BHQ1, BHQ2, BHQ3, BBQ650, ATTO 540Q, ATTO 580Q, ATTO 612Q, CPQ2, QSY-21, QSY-35, QSY-7, QSY-9, DABCYL (4-([4'-dimethylamino)phenyl]azo)benzoyl), Dnp (2,4-dinitrophenyl), or Eclipse®.

[0062] In some embodiments, the fluorescence quencher comprises a BHQ quencher, including but not limited to BHQ0, BHQ1, BHQ2, BHQ3, or BBQ650. BHQ, or black hole quencher, dyes function by a combination of FRET and static quenching, allowing for the avoidance of residual background signals or low signal-to-noise ratios common to fluorescence quenchers such as TAMRA. Different types of BHQ dyes are used to quench dyes of different colors, with BHQ1 being used to quench green and yellow dyes such as FAM, TET, or HEX, and BHQ2 being used to quench orange and red dyes. BHQ dyes are true dark quenchers with no native emission due to their polyachromatic-azo backbone. Substituting electron donating and withdrawing groups on the aromatic ring produces a complete series of quenchers with broad absorption curves spanning the visible spectrum.

[0063] In some embodiments, the fluorescence quencher comprises an ATTO quencher, including but not limited to ATTO 540Q, ATTO 580Q, or ATTO 612Q. The ATTO quencher has the characteristic properties of strong absorption (high extinction coefficient) and high photostability. The ATTO quencher is often utilized as a fluorescence quencher on amine-labeled nucleotides for FRET experiments.

[0064] In some embodiments, the fluorescent quencher comprises CPQ2. The quencher CPQ2 is often used as a pair with the fluorescent donor 5-carboxylfluorescein.

[0065] In some embodiments, the fluorescence quencher comprises a QSY quencher, including but not limited to QSY-21, QSY-35, QSY-7, or QSY-9. QSY probes are dark quenchers, which are substances that absorb excitation energy from a fluorophore and dissipate the energy as heat.

[0066] In some embodiments, the fluorescence quencher comprises DABCYL (4-([4'-dimethylamino)phenyl]azo)benzoyl). DABCYL is one of the most common acceptors for developing FRET-based nucleic acid probes and protease substrates. DABCYL dyes are often paired with EDANS in FRET-based fluorescent probes. DABCYL has broad and strong visible absorption but no fluorescence.

[0067] In some embodiments, the fluorescence quencher comprises Dnp (2,4-dinitrophenyl). Dnp is a stable quencher whose absorption spectrum does not change with pH, ​​making this group a convenient marker for quantitation of substrates in solution.

[0068] In some embodiments, the fluorescence quencher comprises Eclipse®. Eclipse® is a non-fluorescent chromophore and dark quencher that is often used in dual-labeled probes. As a dark quencher, Eclipse® absorbs energy without emitting fluorescence. Eclipse® has an absorption range of 390 nm to 625 nm, allowing it to perform effectively in a wide range of colored FRET probes.

[0069] Carrier In some aspects, the probes / molecules described herein include a carrier. The fluorescence quenchers described herein include any structure. In some embodiments, the carrier includes natural, labeled, or synthetic proteins, synthetic chemical polymers of precisely known chemical composition or with a distribution around an average molecular weight (e.g., linear or branched PEG polymers), oligonucleotides, phosphorodiamidate morpholino oligomers (PMOs), or foldamers, lipids, lipid micelles, nanoparticles (e.g., iron oxide, gold, and non-metallic nanoparticles), solid supports made of polystyrene, polypropylene, or any other type of plastic or polymer. In some embodiments, the carrier includes a peptide that is longer than the peptide linker. The carrier can be covalently or non-covalently bound to the cleavable linker.

[0070] In some embodiments, the carrier comprises a nanoparticle. The transport of insoluble drugs through nanoparticles is improved due to their small particle size. Nanoparticle carriers are a type of sub-microparticle delivery system that belongs to the nanoscale microscope. Drugs encapsulated in subparticles can adjust the rate of drug release, increase the permeability of biofilms, change the distribution in vivo, and improve bioavailability. Nanoparticles are solid colloidal particles in the size range of 10-100 nm that are used as the core of functionalized systems. They are generally composed of natural or synthetic polymeric substances and can be used as carriers to conduct or transport drugs. Nanospheres and nanocapsules can be formed. Chemical materials of nanomaterials are chitosan, gelatin, branched polymers, carbon-based carriers, etc. Gold nanoparticles consist of a core of gold atoms that can be functionalized by the addition of a monolayer of moieties containing thiol (SH) groups.

[0071] In some embodiments, the carrier comprises a natural, labeled, or synthetic protein. Proteins can be used as carriers for the delivery of chemical and biomolecule drugs, such as anti-cancer drugs and therapeutic proteins. Protein nanoparticles have several advantages as drug delivery systems, such as biodegradability, stability, surface modification of particles, ease of particle size control, and fewer issues related to toxicity issues, such as immunogenicity. Protein nanoparticles can be produced using proteins such as fibroin, albumin, gelatin, gliadin, legumin, 30Kc19, lipoprotein, and ferritin proteins, and are prepared by emulsion, electrospray, and desolvation methods. Hong S, Choi DW, Kim HN, Park CG, Lee W, Park HH. Protein-Based Nanoparticles as Drug Delivery Systems. Pharmaceutics. 2020;12(7):604. Published 2020 Jun 29. For example, albumin, a plasma protein with a molecular weight of 66 kDa, has been widely studied as a drug carrier.

[0072] In some embodiments, the carrier comprises a synthetic chemical polymer. Polymeric nanoparticles have been widely studied as drug nanocarriers. Drug loading is achieved either by (i) using the polymer to entrap the aqueous drug phase to form nanoscale structures such as cages and capsules, or (ii) chemically linking drug molecules to the polymer backbone by means of simple ester or amide bonds that can be hydrolyzed in vivo. The most widely studied synthetic polymers include polylactide (PLA), poly(D,L-lactide-co-glycolide) (PLGA), and PEG. All three polymers are hydrolyzed in vivo and biodegradable. Malam Y, Loizidou M, Seifalian AM. Liposomes and nanoparticles: nanosized vehicles for drug delivery in cancer. Trends Pharmacol Sci. 2009 Nov;30(11):592-9.

[0073] In some embodiments, the carrier comprises polyethylene glycol (PEG). PEG has been comprehensively studied as a carrier because it is soluble in both organic and hydrophilic solvents. Unlike many other synthetic polymers, PEG is relatively hydrophilic. In some embodiments, PEG comprises PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, or PEG-8. Conjugation with PEG increases the solubility of hydrophobic molecules and extends their circulation time in the organism. PEG also minimizes non-specific absorption of molecules such as drugs, provides specific affinity for targeted tissues, and increases drug accumulation in malignant tissues. PEG can be conjugated to other polymers to reduce their hydrophobicity (i.e., PEGylation). The change in surface hydrophilicity prevents protein adsorption, thereby allowing cell attachment and proliferation on the biomaterial scaffold. PMO backbones are made of morpholino rings with phosphorodiamidate bonds, which protect them from nuclease degradation while still maintaining complementary base pairing. Potential applications of PMO-based antisense technology targeting bacterial pathogens are being explored for the development of a new class of antibacterial drugs. Panchal RG, Geller BL, Mellbye B, Lane D, Iversen PL, Bavari S. Peptide conjugated phosphorodiamidate morpholino oligomers increase survival of mice challenged with Ames Bacillus anthracis. Nucleic Acid Ther. 2012;22(5):316-322. Fluorescein-tagged morpholinos in combination with fluorescein-specific antibodies can be used as probes for in situ hybridization to miRNAs.

[0074] In some embodiments, the carrier comprises an oligonucleotide. Biostable, high-payload DNA nanoassemblies of various structures have been reported, including cage-like DNA nanostructures, DNA particles, DNA polypods, and DNA hydrogels. The cage-like DNA structures hold drug molecules tightly within the structure, leaving large spaces in the cavity. These DNA nanostructures use their unique structure to carry abundant CpGs and use their biocompatibility and size advantages to enter immune cells and achieve immunotherapy for various diseases. Some of the DNA nanostructures can also be combined with other functional components, such as aPD1, RNA, and TLR ligands, to achieve more effective treatment. DNA-based nanoparticles, such as spherical nucleic acids, hybrid DNA-based nanoparticles, polypod-like DNA nanostructures, and DNA hydrogels, have been reported. Chi Q, Yang Z, Xu K, Wang C and Liang H(2020)DNA Nanostructure as an Efficient Drug Delivery Platform for Immunotherapy.Front.Pharmacol.10:1585.

[0075] In some embodiments, the carrier comprises a phosphorodiamidate morpholino oligomer (PMO). Antisense phosphorodiamidate morpholino oligomers (PMOs) and their derivatives down-regulate target gene expression in a sequence-dependent manner by interfering with ribosome binding to mRNA, thereby inhibiting protein translation.

[0076] In some embodiments, the carrier comprises a lipid or lipid micelle. The liposome bilayer can be composed of either synthetic or natural phospholipids. The primary physical and chemical properties of liposomes are based on the net properties of the constituent phospholipids, including permeability, charge density, and steric hindrance. The lipid bilayer closes on itself due to interactions between water molecules and the hydrophobic phosphate groups of the phospholipids. This process of liposome formation is spontaneous, as amphipathic phospholipids self-associate into the bilayer. Drug loading into liposomes can be achieved by (i) liposome formation in aqueous solutions saturated with soluble drugs, (ii) use of organic solvents and solvent exchange mechanisms, (iii) use of lipophilic drugs, and (iv) pH gradient methods. Malam Y, Loizidou M, Seifalian AM. Liposomes and nanoparticles: nanosized vehicles for drug delivery in cancer. Trends Pharmacol Sci. 2009 Nov;30(11):592-9.

[0077] In some embodiments, the carrier comprises a solid support made of polystyrene, polypropylene, or any other type of plastic. For example, drug delivery properties of microporous polystyrene solid foams have been reported by Canal et al. These materials were obtained by polymerization in the continuous phase of highly concentrated emulsions prepared by the phase inversion temperature method. Their porosity, specific surface area, and surface topography are related to drug incorporation and release properties. Canal, Cristina & Aparicio, Rosa & Vilchez, Alejandro & Esquena, Jordi & Garcia-Celma, Maria. (2012). Drug Delivery Properties of Macroporous Polystyrene Solid Foams. Journal of pharmacy & pharmaceutical sciences: a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques. 15. 197-207.

[0078] In some embodiments, the carrier comprises a foldamer. A foldamer is a folded oligomer or polymer with a well-defined conformation. Because the conformation of foldamers is highly predictable from their primary sequence, it is possible to place functional groups at target positions, e.g., to design functional foldamers for efficient cellular uptake. For example, cell-penetrating peptide (CPP) foldamers are peptide-based foldamers with cell membrane permeability. Peptide foldamers contain unnatural amino acids, non-proteinogenic amino acids, which allow the peptides to adopt stable secondary structures, especially helical structures, even with short sequences. This property is useful for the design of amphipathic CPPs with stable helical structures. Furthermore, peptides containing unnatural amino acids generally exhibit resistance to hydrolysis by proteases that are abundant throughout the body and in cells. The high stability of peptide foldamers against enzymatic degradation can lead to their long-term function in vivo. Makoto Oba,Cell-Penetrating Peptide Foldamers:Drug Delivery Tools.ChemBioChem 10.1002 / cbic.201900204.

[0079] Self-Sacrificing Spacer In some aspects, the probes / molecules described herein include a self-immolative spacer. In some embodiments, the self-immolative spacer includes a disulfide, p-aminobenzyl alcohol, a-quinone methide spacer, heteroamine bifunctional disulfide, thiol-based pyridazinedione, p-amine benzyloxycarbonyl, dipeptide, Gly-Pro (SEQ ID NO: 530), L-Phe-Sar, trans-cyclooctenetetrazine, ortho-hydroxy-protected aryl sulfate, phosphoramidate-based spacer, hydroxybenzyl, trimethylcarbamate, quinone methide-based spacer, cyclization spacer, trimethyl lock, 2-aminomethylpiperidine, or ethylenediamine-derived cyclization spacer. Gonzaga et al. Perspective about self-immolative drug delivery systems. Journal of Pharmaceutical Sciences 109(2020)3262-3281.

[0080] Cleavage of the cleavable linker by a predetermined protease or enzyme dissociates the self-immolative spacer from the precipitating fluorescent or non-fluorescent reporter, thereby resulting in a detectable signal. The cleavable linkers of the multiple probes / molecules are cleavable by a predetermined endoprotease in the bodily fluid sample, resulting in self-immolation and reporter release, or provide a protease substrate that can be cleaved by a predetermined exopeptidase. In some embodiments, a predetermined exopeptidase is added to the bodily fluid sample. In some embodiments, the predetermined exopeptidase cleaves the protease substrate, thereby dissociating the self-immolative spacer from the precipitating fluorescent reporter, thereby resulting in a detectable signal.

[0081] Body fluid samples The determination of the disease or condition is based on the rate of formation or amount of the released reporter detected in the bodily fluid sample. In some embodiments, the bodily fluid sample includes blood, serum, plasma, bone marrow fluid, lymphatic fluid, bile, amniotic fluid, mucosal fluid, saliva, urine, cerebrospinal fluid, synovial fluid, semen, ductal aspirate, stool, vaginal effluent, cyst fluid, tissue homogenate, tissue-derived fluid, tears, and patient-derived cell line supernatant. In some embodiments, the bodily fluid sample includes rinses. In some embodiments, rinses include mouthwash rinses, bronchoalveolar rinses, lavage fluids, hairwash rinses, nasal spray effluents, swabs of any body surface, orifice, or organ structure applied to saline or any medium, or any derivative thereof.

[0082] In some embodiments, the body fluid sample comprises blood. Blood is a constantly circulating fluid that provides the body with nutrients, oxygen, and waste removal. Blood is primarily liquid with a large number of cells and proteins suspended within it. Blood is made up of several major components including plasma, red blood cells, white blood cells, and platelets.

[0083] In some embodiments, the body fluid sample comprises plasma. Plasma is the liquid remaining when clotting is prevented with the addition of an anticoagulant. Serum is the conventional term in the art for the fluid remaining when clotting factors are removed from plasma. Anticoagulants are drugs that help prevent blood clots. Examples of anticoagulants include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citrate, heparin, oxalate, any salts, solvates, enantiomers, tautomers, and geometric isomers thereof, or any mixtures thereof.

[0084] In some embodiments, the bodily fluid sample includes bone marrow fluid. Bone marrow is found in the center of most bones and has many blood vessels. There are two types of bone marrow: red and yellow. Red bone marrow contains blood stem cells that can become red blood cells, white blood cells, or platelets. Yellow bone marrow is made up primarily of fat.

[0085] In some embodiments, the body fluid sample comprises lymph, also called lymph, which is the collection of excess fluid that is drained from cells and tissues and is not reabsorbed into the capillaries.

[0086] In some embodiments, the bodily fluid sample comprises bile. Bile is a digestive fluid produced by the liver and stored in the gallbladder. During bile reflux, digestive fluids return to the stomach and, in some cases, the esophagus.

[0087] In some embodiments, the bodily fluid sample comprises amniotic fluid, which is the clear, slightly yellowish fluid that surrounds an unborn baby (fetus) during pregnancy. It is contained in the amniotic sac.

[0088] In some embodiments, the bodily fluid sample comprises mucosal fluid. Mucosal fluid, also called mucus, is a thick, protective fluid secreted by mucous membranes and used to keep pathogens and dirt out of the body. Mucus is also used to keep bodily tissues from dehydrating.

[0089] In some embodiments, the bodily fluid sample comprises saliva, which is an extracellular fluid produced and secreted by the salivary glands in the mouth.

[0090] In some embodiments, the bodily fluid sample comprises urine, which is the liquid by-product of metabolism in humans and many other animals. Urine flows from the kidneys through the ureters to the bladder.

[0091] In some embodiments, the bodily fluid sample comprises cerebrospinal fluid, a clear fluid that surrounds the brain and spinal cord. It cushions the brain and spinal cord from injury and also serves as a nutrient delivery and waste removal system for the brain.

[0092] In some embodiments, the fluid sample comprises synovial fluid. Synovial fluid, also known as joint fluid, is a thick liquid located between joints. The fluid cushions the ends of bones and reduces friction when the joint moves.

[0093] In some embodiments, the bodily fluid sample comprises semen, which is a male reproductive fluid that contains sperm in suspension.

[0094] In some embodiments, the fluid sample comprises a ductal aspirate. Ductal aspirate, also known as ductal lavage, ductal fluid, or lavage fluid, is fluid collected from a duct, such as a milk duct in the breast.

[0095] In some embodiments, the bodily fluid sample comprises stool. Stool, also known as excrement or feces, is waste material that leaves the intestines after food is digested.

[0096] In some embodiments, the bodily fluid sample can be vaginal flow. Vaginal flow, also known as vaginal secretion, is a clear or whitish fluid that comes from the vagina.

[0097] In some embodiments, the bodily fluid sample can be tears, which is secreted by the tear gland to lubricate the eye and fight bacteria.

[0098] In some embodiments, the body fluid sample comprises a tissue homogenate, which is obtained by mechanical microdisruption of fresh tissue, and the cell membranes are mechanically permeabilized.

[0099] Proteases and other drugs The probes / molecules described herein can be cleaved by proteases from bodily fluids. In some embodiments, the proteases include endopeptidases or exopeptidases.

[0100] In some embodiments, the protease comprises an endopeptidase, which is an enzyme that breaks peptide bonds other than the terminal bonds in a peptide chain.

[0101] In some embodiments, the protease comprises an exopeptidase, an enzyme that catalyzes the cleavage of terminal or penultimate peptide bonds, a process that releases single amino acids or dipeptides from the peptide chain.

[0102] In some embodiments, the protease is A20 (TNFα-induced protein 3), Abhydrolase domain-containing 4, Abhydrolase domain-containing 12, Abhydrolase domain-containing 12B, Abhydrolase domain-containing 13, acrosin, acylaminoacyl-peptidase, a disintegrin and metalloproteinase (ADAM), ADAM1a, ADAM2 (fertilin-b), ADAM3B, ADAM4, ADAM4B, ADAM5, ADAM6, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADA M12 metalloproteases, ADAM15, ADAM17, ADAM18, ADAM19, ADAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM32, ADAM33, A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS4, ADAMTS5 / 11, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS 13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20, adipocyte-associated protein 1, Afg3-like protein 1, Afg3-like protein 2, airway trypsin-like protease, aminoacylase, aminopeptidase A, aminopeptidase B, aminopeptidase B-like 1, aminopeptidase MAMS / L-RAP, aminopeptidase N, aminopeptidase O, aminopeptidase P homolog, aminopeptidase P1, aminopeptidase PILS, aminopeptidase putidase Q, aminopeptidase-like 1, AMSH / STAMBP, AMSH-LP / STAMBPL1, angiotensin-converting enzyme 1 (ACE1), angiotensin-converting enzyme 2 (ACE2), angiotensin-converting enzyme 3 (ACE3), anionic trypsin(II), apolipoprotein(a), archaemetzincin-1, archaemetzincin-2, aspartoacylase, aspartoacylase-3, aspartyl aminopeptidase, ataxin-3, ataxin-3-like, ATP / GTP-binding protein 1,ATP / GTP-binding protein-like 2, ATP / GTP-binding protein-like 3, ATP / GTP-binding protein-like 4, ATP / GTP-binding protein-like 5, ATP23 peptidase, autophagin-1, autophagin-2, autophagin-3, autophagin-4, azurocidin, or a combination thereof.

[0103] In some embodiments, the protease comprises beta-lactamase, beta-secretase 1, beta-secretase 2, bleomycin hydrolase, brain serine proteinase 2, BRCC36 (BRCA2 containing complex, sub3), calpain, calpain 1, calpain 2, calpain 3, calpain 4, calpain 5, calpain 6, calpain 7, calpain 7-like, calpain 8, calpain 9, calpain 10, calpain 11, calpain 12, calpain 13, calpain 14, calpain 15 (Solh proteins), or a combination thereof.

[0104] In some embodiments, the protease is a cysteine ​​protease, carboxypeptidase A1, carboxypeptidase A2, carboxypeptidase A3, carboxypeptidase A4, carboxypeptidase A5, carboxypeptidase A6, carboxypeptidase B, carboxypeptidase D, carboxypeptidase E, carboxypeptidase M, carboxypeptidase N, carboxypeptidase O, carboxypeptidase U, carboxypeptidase X1, carboxypeptidase X2, Carboxypeptidase Z, carnosin dipeptidase 1, carnosin dipeptidase 2, caspase recruitment domain family, member 8, caspase, caspase-1, caspase-2, caspase-3, caspase-4 / 11, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-12, caspase-14, caspase-14-like, caspase / FLIP, cathepsin, cathepsin A (CTSA), cathepsin B (CTSB), cathepsin C (CTSC), cathepsin A (CTSA), cathepsin B (CTSB), cathepsin C (CTSC), cathepsin B ... Cathepsin D (CTSD), cathepsin E (CTSE), cathepsin F, cathepsin G, cathepsin H (CTSH), cathepsin K (CTSK), cathepsin L (CTSL), cathepsin L2, cathepsin O, cathepsin S (CTSS), cathepsin V (CTSV), cathepsin W, cathepsin Z (CTSZ), cationic trypsin, Cezanne / OTU domain-containing 7B, Cezanne-2, CGI-58, chymase, chymopathin, chymosin, chymotrypsin B, chymotrypsin C, coagulation factor IXa, coagulation factor VIIa, coagulation factor and / or combinations thereof.

[0105] In some embodiments, the protease is a DDI-related protease, DECYSIN, Der1-like domain family, member 1, Der1-like domain family, member 2, Der1-like domain family, member 3, DESC1 protease, desert hedgehog protein, desumoylated isopeptidase 1, desumoylated isopeptidase 2, dihydroorotase, dihydropyrimidinase, dihydropyrimidinase-related protein 1, dihydropyrimidinase-related protein 2, dihydropyrimidinase-related protein 3, dihydropyrimidinase-related protein 4, dihydropyrimidinase-related protein 5, dihydropyrimidinase-related protein 6, dihydropyrimidinase-related protein 7, dihydropyrimidinase-related protein 8, dihydropyrimidinase-related protein 9, dihydropyrimidinase-related protein 10, dihydropyrimidinase-related protein 11, dihydropyrimidinase-related protein 12, dihydropyrimidinase-related protein 13, dihydropyrimidinase-related protein 14, dihydropyrimidinase-related protein 15, dihydropyrimidinase-related protein 16, dihydropyrimidinase-related protein 17, dihydropyrimidinase-related protein 18, dihydropyrimidinase-related protein 19, dihydropyrimidinase-related protein 20, dihydropyrimidinase-related protein 21, dihydropyrimidinase-related protein 22, dihydropyrimidinase-related protein 23, dihydropyrimidinase-related protein 24, dihydropyrimidinase-related protein 19, dihydropyrimidinase-related protein 19, dihydropyrimidinase-related protein 25, dihydropyrimidinase-related protein 26, dihydropyrimidinase-related protein 27, dihydropy and / or combinations thereof.

[0106] In some embodiments, the protease comprises enamelysin, endopeptidase Clp, endoplasmic reticulum metallopeptidase 1, endothelin-converting enzyme 1, endothelin-converting enzyme 2, enteropeptidase, epidermis-specific SP-like, epilysin, epithelial cell transforming sequence 2 oncogene-like, epitheliacin, epoxide hydrolase, epoxide hydrolase-related protein, eukaryotic translation initiation F3SF, eukaryotic translation initiation F3SH, or a combination thereof.

[0107] In some embodiments, the protease comprises Factor VII activating protease, FACE-1 / ZMPSTE24, FACE-2 / RCE1, family 108 with sequence similarity, member A1, family 108 with sequence similarity, member B1, family 108 with sequence similarity, member C1, family 111 with sequence similarity, A, family 111 with sequence similarity, B, furin, or a combination thereof.

[0108] In some embodiments, the protease is gamma-glutamyl hydrolase, gamma-glutamyl transferase 1, gamma-glutamyl transferase 2, gamma-glutamyl transferase 5, gamma-glutamyl transferase 6, gamma-glutamyl transferase m-3, gamma-glutamyl transferase-like 3, GCDFP15, gelatinase A, gelatinase B, Gln-fructose-6-P transamidase 1, Gln- fructose-6-P transamidase 2, Gln-fructose-6-P transamidase 3, Gln-PRPP amidotransferase, glutamate carboxypeptidase II, glutaminyl cyclase, glutaminyl cyclase 2, glycosylasparaginase, glycosylasparaginase-2, granzyme, granzyme A, granzyme B, granzyme H, granzyme K, granzyme M, haptoglobin-1, or a combination thereof.

[0109] In some embodiments, the protease is histone deacetylase (HDAC), haptoglobin-associated protein, HAT-like 2, HAT-like 3, HAT-like 4, HAT-like 5, HAT-associated protease, HSP90AA1? (heat shock 90 kDa protein 1, alpha), HSP90AB1? (heat shock 90 kDa protein 1, beta), heat shock protein 75, heat shock protein 90 kDa beta (Grp94), member 1 / tumor rejection antigen (Glp95), ... beta), HSP90AA1? (heat shock 90 kDa protein 1, beta), HSP90AA1? (heat shock 90 kDa protein 1, beta), HSP90AB1? (heat shock 90 kDa protein 1, beta), heat shock protein 75, heat shock protein 90 kDa beta (Grp94), member 1 / tumor rejection antigen (Glp95), HSP90AA1? (heat shock 90 kDa protein 1, beta), HSP90AA1? (heat shock 90 kDa protein 1, beta), HSP90AB1? (heat shock 90 kDa protein 1, beta), HSP90AA1? (heat shock 90 kDa protein 1, beta), HSP90AA1? (heat shock 90 kDa protein 1, beta), HSP90AB1? (heat shock 90 kDa protein 1, beta), HSP p96), hepatocyte growth factor, hepsin, HetF-like, HGF activator, hGPI8, Hin-1 / OTU domain containing 4, homolog ICEY, HP43.8KD, HTRA1 serine protease, HTRA2, HTRA3, HTRA4, hyaluronan-binding ser-protease, transplanted serine protease 2, Indian hedgehog protein, insulysin, intestinal serine protease 1, Josephin-1, Josephin-2, or a combination thereof.

[0110] In some embodiments, the protease comprises kallikrein (KLK), kallikrein hK1, kallikrein hK2, kallikrein hK3, kallikrein hK4, kallikrein hK5, kallikrein hK6, kallikrein hK7, kallikrein hK8, kallikrein hK9, kallikrein hK10, kallikrein hK11, kallikrein hK12, kallikrein hK13, kallikrein hK14, kallikrein hK15, Kell blood group protein, KHNYN KH and NYN domain containing, lactotransferrin, legumain, leissima nolysin-2, leucyl aminopeptidase, leucyl-cystinyl aminopeptidase, leukotriene A4 hydrolase, lysosomal carboxypeptidase A, lysosomal pro-X C-peptidase, or a combination thereof.

[0111] In some embodiments, the protease is membrane metallo-endopeptidase (MME), macrophage elastase, macrophage stimulating protein, mammalian tolloid-like 1 protein, mammalian tolloid-like 2 protein, MAP1D methionine aminopeptidase 1D, marapsin, marapsin 2, MASP1 / 3 (MBL-associated serine protease 3), MBL-associated serine protease 2 (MASP2), mustin, matrilysin, matrilysin-2, matriptase, matriptase-2, matriptase-3, membrane dipeptidase, membrane dipeptidase 2, membrane dipeptidase 3, membrane mosaic Ser-protein, meprin alpha subunit, meprin beta peptidase b-subunit, mitochondrial proc. protease, mitochondrial signal peptidase, matrix metalloproteinase (MMP), MMP19, MMP21, MMP23A, MMP23B, MMP27, MPND, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, MYSM1, or a combination thereof.

[0112] In some embodiments, the protease is NAALADASE II, NAALADASE-like 2, NAALADASE-like 1, napsin A, napsin B, nardilysin, nasal embryonic LHRH factor, NEDD4 binding protein 1, neprilysin, neprilysin-2, neurolysin, neurotrypsin, neutrophil elastase (ELANE, ELA2), NLRP1 autocleaving protein, nuclear receptor interacting protein 2, nuclear receptor interacting protein 3, nucleoporin 98, NYN domain, and retroviral integrase. containing, NY-REN-60, OMA1, O-sialoglycoprotein endopeptidase, O-sialoglycoprotein endopeptidase-like 1, osteoblast serine protease, OTU domain-containing 6B, OTU domain-containing-1, OTU domain-containing-3, OTU domain-containing-5, OTU domain-containing-6A, otubain-1, otubain-2, OTUD2 / YOD1, ovastacin, oviductin-like / ovochymase-2, or a combination thereof.

[0113] In some embodiments, the protease is proteinase 3 (PRTN3), papain, PACE4 proprotein convertase, pancreatic elastase, pancreatic elastase II (IIA), pancreatic elastase II type B, pancreatic endopeptidase E (A), pancreatic endopeptidase E (B), papalysin-1, papalysin-2, paracaspase, paraplesin, pepsin A, pepsin C, PHEX endopeptidase, PIDD autoprocessing protein unit 1, PIM1 endopeptidase, PIM2 endopeptidase, pitrilysin metalloproteinase, inase 1, plasma Glu-carboxypeptidase, plasma kallikrein, plasma kallikrein-like 2, plasma kallikrein-like 3, plasma kallikrein-like 4, plasmin (plasminogen), PM20D2 peptidase, POH1 / PSMD14, polyserase-2, polyserase-3, polyserase-I, Ppnx, presenilin 1, presenilin 2, presenilin homolog 1 / SPPL3, presenilin homolog 2, presenilin homolog 3 / SPP, presenilin homolog 4 / SPPL2B, presenilin homolog 5, presenilin-associated rhomboid-like, procollagen C proteinase, proliferation-associated protein 1, prolyl oligopeptidase, proligopeptidase-like, proprotein convertase 1, proprotein convertase 2, proprotein convertase 4, proprotein convertase 5, proprotein convertase 7, proprotein convertase 9 (proprotein convertase subtilisin / kexin type 9, PCSK9), prostasin, (protease, serine, 56), proteasome alpha 1 subunit, proteasome alpha 2 subunit, proteasome alpha 3 subunit, proteasome alpha 3-like subunit, proteasome alpha 4 subunit, proteasome alpha 5 subunit, proteasome alpha 6 subunit, proteasome alpha 7 subunit, proteasome alpha 8 subunit, proteasome b subunit LMP7-like, proteasome beta 1 subunit, proteasome beta 2 subunit, proteasome beta 3 subunit, proteasome beta 3-like subunit, proteasome beta 4 subunit, proteasome catalytic subunit 1-like, proteasome catalytic subunit 1,proteasome catalytic subunit 1i, proteasome catalytic subunit 2, proteasome catalytic subunit 2i, proteasome catalytic subunit 3, proteasome catalytic subunit 3i, protein C, protein C-like, protein Z, proteinase 3, PRPF8, PSMD7, pyroglutamyl-peptidase I, pyroglutamyl-peptidase II, or a combination thereof.

[0114] In some embodiments, the protease comprises reelin, renin, retinol binding protein 3, rhomboid 5 homolog 1, rhomboid 5 homolog 2, rhomboid domain-containing 1, rhomboid domain-containing 2, rhomboid, venule-like 2, rhomboid, venule-like 3, rhomboid-like protein 1, or a combination thereof.

[0115] In some embodiments, the protease is a serine protease, serine protease 3 (PRSS3), S2P protease, SAD1, cecernin-1, cecernin-2, cecernin-3, centrin (SUMO protease 1), centrin (SUMO protease 2), centrin (SUMO protease 3), centrin (SUMO protease 5), centrin (SUMO protease 5-like 1), centrin (SUMO protease 6), centrin (SUMO protease 7), centrin (SUMO protease 8), centrin (SUMO protease 9), centrin (SUMO protease 11), centrin (SUMO protease 12), centrin (SUMO protease 1 3), sentrin (SUMO protease 14), sentrin (SUMO protease 15), sentrin (SUMO protease 16), sentrin (SUMO protease 17), sentrin (SUMO protease 18), sentrin (SUMO protease 19), separase, seprase, serine carboxypeptidase 1, signalase 18 kDa component, signalase 21 kDa component, signalase-like 1, similar to Arabidopsis Ser-protease, similar to SPUVE, site 1 protease, sonic hedgehog protein, spinesin, SprT-like N-terminal domain, stromelysin 1, stromelysin 2, stromelysin 3, suppressor of Ty16 homolog, or a combination thereof.

[0116] In some embodiments, the protease is taspase, TBP-related factor 2, TESP2, TESP3, testase 2, testicular serine protease 2, testicular serine protease 3, testicular serine protease 4, testicular serine protease 5, testicular serine protease 6, testisin, testis-specific protein tsp50, thimet oligopeptidase, thrombin, thymus-specific serine peptidase, TINAG-related protein, TMPRSS11A, t-plasminogen activator, TRAF-binding protein domain, transferrin receptor 2 protein. transmembrane Ser-protease 4, transthyretin, TRH-degrading ectoenzyme, tripeptidyl-peptidase I, tryptidyl-peptidase II, trypsin, trypsin 10, trypsin 15, trypsin C, trypsin X2, tryptase, tryptase alpha / beta 1, tryptase beta 2, tryptase delta 1, tryptase gamma 1, tryptase homolog 2 / EOS, tryptase homolog 3, tubulointerstitial nephritis antigen, or a combination thereof.

[0117] In some embodiments, the protease is ubiquitin C-terminal hydrolase BAP1, ubiquitin C-terminal hydrolase 1, ubiquitin C-terminal hydrolase 3, ubiquitin C-terminal hydrolase 4, ubiquitin C-terminal hydrolase 5, ubiquitin-specific peptidase-like 1, UCR1, UCR2, UDP-N-acetylglucosaminyltransferase subunit, Ufm-1 specific protease 1, Ufm-1 specific protease 2, urokinase (PLAU, uPA) umbilical vein proteinase, u-plasminogen activator, USP1, USP2, USP3, USP4, USP5, USP6, USP7, US P8, USP9X, USP9Y, USP10, USP11, USP12, USP13, USP14, USP15, USP16, USP17, USP17-like, USP18, USP19, USP20, USP21, USP22, USP24, USP25, USP26, USP27, USP28, USP29, USP30, USP31, USP34, USP35, USP36, USP37, USP40, USP41, USP42, USP43, USP44, USP45, USP46, USP47, USP48, USP49, USP50, USP51, USP52, USP53, USP54, or combinations thereof.

[0118] In some embodiments, the protease comprises VCP(p97) / p47 interacting protein, VDU1, vitellogenic carboxypeptidase-L, X-Pro dipeptidase, X-prolyl aminopeptidase 2, YME1-like 1, zinc finger CCCH type-containing 12A, zinc finger CCCH type-containing 12B, zinc finger CCCH type-containing 12C, zinc finger CCCH type-containing 12D, zinc finger-containing ubiquitin peptidase 1, or a combination thereof.

[0119] In some embodiments, the protease comprises A20 (tumor necrosis factor, alpha-induced protein 3, TNFα-induced protein 3). A20 is a zinc finger protein and a deubiquitinase. A20 has been shown to inhibit NF-kappaB activation and TNF-mediated apoptosis, limiting inflammation.

[0120] In some embodiments, the protease comprises angiotensin-converting enzyme 2 (ACE2). ACE2 is a membrane-bound enzyme located on the membrane of cells located in the intestine, kidney, testes, gallbladder, and heart. ACE2 counteracts the activity of the related angiotensin-converting enzyme, ACE, by reducing the amount of angiostatin II.

[0121] In some embodiments, the protease comprises a cathepsin. Cathepsins can be, but are not limited to, cathepsin A (CTSA), cathepsin B (CTSB), cathepsin C (CTSC), cathepsin D (CTSD), cathepsin E (CTSE), cathepsin H (CTSH), cathepsin K (CTSK), cathepsin L (CTSL), cathepsin S (CTSS), cathepsin V (CTSV), and cathepsin Z (CTSZ). Cathepsins are a subset of proteases, many of which are activated at low pH. Cathepsins include, among others, serine proteases, cysteine ​​proteases, and aspartyl proteases. Cathepsins are involved in cancer, Alzheimer's disease, arthritis, Ebola, pancreatitis, glaucoma, COPD, and other diseases.

[0122] In some embodiments, the protease comprises a caspase, which can be, but is not limited to, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, and caspase 14.

[0123] In some embodiments, the protease comprises a calpain, which can be, but is not limited to, calpain 1, calpain 2, calpain 3, calpain 4, calpain 5, calpain 6, calpain 7, calpain 8, calpain 9, calpain 10, calpain 11, calpain 12, calpain 13, calpain 14, and calpain 15. Caspases are a family of protease enzymes that play essential roles in programmed cell death and cellular homeostasis.

[0124] In some embodiments, the protease comprises a cysteine ​​protease. Cysteine ​​proteases, also known as thiol proteases, are hydrolase enzymes that degrade proteins. These proteases share a common catalytic mechanism that involves a nucleophilic cysteine ​​thiol in the catalytic triad or dyad. The cysteine ​​protease family includes papain (Carica papaya), bromelain (Ananas comosus), cathepsin K (Liverwort), calpain (Homo sapiens), aspase-1 (Rattus norvegicus), separase (Saccharomyces cerevisiae), adenine (human adenovirus type 2), pyroglutamyl-peptidase I (Bacillus amyloliquefaciens), sortase A (Staphylococcus aureus), hepatitis C virus peptidase 2 (hepatitis C virus), Sindbis virus nsP2 type peptidase (Sindbis virus), dipeptidyl-peptidase VI (Lysinibacillus sphaericus), DeSI-1 peptidase (Mus musculus), TEV protease (tobacco etch virus), amidophosphoribosyltransferase precursor (Homo sapiens), gamma-glutamyl hydrolase (Rattus norvegicus), hedgehog protein (Drosophila melanogaster), and DmpA aminopeptidase (Ochrobactrum anthropi).

[0125] In some embodiments, the protease comprises complement C1r serine protease (complement component 1r). In some embodiments, the protease comprises complement C1s serine protease (complement component 1s). C1r forms the C1 complex with C1q and C1s. C1r has a very narrow trypsin-like specificity that is involved in the activation of the C1 complex. C1 activation is a two-step process involving (1) C1r intramolecular autoactivation and (2) C1s cleavage by activated C1r. C1r contains a chymotrypsin-like serine protease domain at its C-terminus that cleaves a single Arg-Ile bond in C1r and C1s. Zvi Fishelson, in xPharm: The Comprehensive Pharmacology Reference, 2007.

[0126] In some embodiments, the protease comprises chymotrypsin (chymotrypsin A and B, alpha-chymalofus, abaszyme, chymal, chymotest, enzeon, chymal, chymotolase, alpha-chymal, alpha-chymotrypsin A, alpha-chymotrypsin). Chymotrypsin is a digestive enzyme component of pancreatic juice that acts in the duodenum to perform proteolysis, the breakdown of proteins and polypeptides. Chymotrypsin preferentially cleaves peptide amide bonds, and the side chains of amino acids N-terminal to the scissile amide bond are large hydrophobic amino acids (tyrosine, tryptophan, and phenylalanine).

[0127] In some embodiments, the protease comprises chymase (mast cell protease 1, skeletal muscle protease, skin chymotrypsin proteinase, mast cell serine proteinase, skeletal muscle protease). Chymase is a family of serine proteases found in mast cells, basophil granulocytes. Chymase exhibits a wide range of peptidolytic activity and is involved in inflammatory responses, hypertension, and atherosclerosis.

[0128] In some embodiments, the protease comprises a dipeptidyl peptidase (DPP). DPPs include cathepsin C (DPP1), DPP2, DPP3, DPP4, DPP6, DPP7, DPP8, DPP9, and DPP10.

[0129] In some embodiments, the protease comprises DPP4 (adenosine deaminase complex forming protein 2, CD26). DPP4 is expressed on the cell surface and is associated with immune regulation, signal transduction, and apoptosis. DPP4 is a serine exopeptidase that cleaves X-proline or X-alanine dipeptides from the N-terminus of polypeptides. DPP-4 is known to cleave a wide range of substrates, including growth factors, chemokines, neuropeptides, and vasoactive peptides. DPP4 plays a key role in glucose metabolism, is involved in the degradation of incretins such as GLP-1, and appears to function as an inhibitor in the development of some tumors.

[0130] In some embodiments, the protease comprises DPP1 (cathepsin C, CTSC). DPP1 is a lysosomal exocysteine ​​protease belonging to the peptidase C1 family. Cathepsin C appears to be a central regulator for the activation of many serine proteases in immune / inflammatory cells. Cathepsin C catalyzes the excision of dipeptides from the N-terminus of protein and peptide substrates.

[0131] In some embodiments, the protease comprises a disintegrin and metalloproteinase (ADAM). ADAMs are a family of single-pass transmembrane and secreted metalloendopeptidases. Not all human ADAMs have a functional protease domain. Those ADAMs that are active proteases are classified as sheddases because they clip or remove the extracellular portion of transmembrane proteins.

[0132] In some embodiments, the protease comprises the ADAM12 metalloprotease, which binds to insulin growth factor binding protein-3 (IGFBP-3), appears to be an early Down's syndrome marker, and is involved in a variety of biological processes involving cell-cell and cell-matrix interactions, including fertilization, muscle development, and neurogenesis.

[0133] In some embodiments, the protease comprises a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS). ADAMTS is a family of multi-domain extracellular protease enzymes, including ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS4, ADAMTS5 (=ADAMTS11), ADAMTS6, ADAMTS7, ADAMTS8 (or METH-2), ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, and ADAMTS20. Known functions of ADAMTS proteases include processing of procollagen and von Willebrand factor, as well as cleavage of aggrecan, versican, brevican, and neurocan, making them key remodeling enzymes of the extracellular matrix. They have been shown to have important roles in connective tissue organization, coagulation, inflammation, arthritis, angiogenesis, and cell migration.

[0134] In some embodiments, the protease comprises ADAMTS1. ADAMTS1 is a member of the ADAMTS protein family. The expression of ADAMTS1 can be associated with various inflammatory processes, the development of cancer cachexia, normal growth, fertility, and organ morphology and function.

[0135] In some embodiments, the protease comprises factor VII activating protease (FSAP), a circulating serine protease with high homology to fibrinolytic enzymes and may be involved in the regulation of coagulation and fibrinolysis.

[0136] In some embodiments, the protease comprises furin, which belongs to the subtilisin-like proprotein convertase family and is a calcium-dependent serine endoprotease. Substrates of furin include parathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type 1 matrix metalloproteinase, beta subunit of pro-nerve growth factor, and von Willebrand factor.

[0137] In some embodiments, the protease comprises a histone deacetylase (HDAC), a class of enzymes that remove acetyl groups (O=C-CH3) from ε-N-acetyl lysine amino acids on histones, allowing the histones to more tightly wrap around DNA.

[0138] In some embodiments, the protease comprises HTRA1 serine protease. HTRA1 is a secreted enzyme that has been shown to regulate insulin-like growth factor (IGF) availability by cleaving IGF-binding proteins. It has also been suggested to be a regulator of cell growth.

[0139] In some embodiments, the protease comprises granzyme. Granzyme is a serine protease released by cytoplasmic granules in cytotoxic T cells and natural killer (NK) cells. Granzyme induces programmed cell death in target cells. Granzyme also kills bacteria and inhibits viral replication.

[0140] In some embodiments, the protease comprises kallikrein (KLK). Kallikreins are a subgroup of serine proteases. Kallikreins are involved in regulating a variety of physiological functions, including blood pressure, semen liquefaction, and skin desquamation.

[0141] In some embodiments, the protease comprises a matrix metalloproteinase (MMP, matrix metallopeptidase, matrixin). MPPs are calcium-dependent zinc-containing endopeptidases. MMPs are involved in cleavage of cell surface receptors, release of apoptotic ligands, chemokine / cytokine inactivation, cell proliferation, and cell migration.

[0142] In some embodiments, the protease comprises membrane metallo-endopeptidase (MME). MME is a zinc-dependent metalloprotease that cleaves peptides at the amino side of hydrophobic residues, inactivating several peptide hormones, including glucagon, enkephalin, substance P, neurotensin, oxytocin, and bradykinin. MME is expressed in a wide variety of tissues and is particularly abundant in the kidney. MME is also a common acute lymphocytic leukemia antigen.

[0143] In some embodiments, the protease comprises mannose-binding protein-associated serine protease 2 (MASP2, mannan-binding lectin serine protease 2, MBL-associated serine protease 2). MASP2 is involved in the complement system and cleaves complement components C4 and C2 into C4a, C4b, C2a, and C2b.

[0144] In some embodiments, the protease comprises mannose-binding protein-associated serine protease 3 (MBL-associated serine protease 3, MASP3), which is derived from the MASP1 gene by differential splicing, circulates at high serum concentrations primarily in complex with ficolin-3, and regulates ficolin-3-mediated complement activation.

[0145] In some embodiments, the protease comprises neutrophil elastase (ELANE, ELA2), a serine proteinase secreted by neutrophils and microphages during inflammation that destroys bacteria and host tissue.

[0146] In some embodiments, the protease comprises protease 3 (PRTN3), a serine protease enzyme expressed primarily in neutrophil granulocytes and which contributes to the proteolytic generation of antimicrobial peptides.

[0147] In some embodiments, the protease comprises plasmin (also known as plasminogen). Plasmin is a proteolytic enzyme derived from an inactive plasma precursor known as plasminogen. It is present in blood and breaks down many plasma proteins, including fibrin clots. In humans, plasmin is encoded by the PLG gene.

[0148] In some embodiments, the protease comprises pepsin. Pepsin is an endopeptidase that cleaves proteins into smaller peptides. It is an aspartic protease that uses a catalytic aspartate in its active site.

[0149] In some embodiments, the protease comprises presenilin-1 (PS-1). PS-1 is a presenilin protein that is one of the four core proteins in the gamma secretase complex, which is believed to play a key role in the generation of amyloid beta from amyloid precursor protein.

[0150] In some embodiments, the protease comprises proprotein convertase subtilisin / kexin type 9 (PCSK9). PCSK9 is a member of the peptidase S8 family.

[0151] In some embodiments, the protease comprises a serine protease. Serine proteases cleave peptide bonds in proteins, with serine functioning as the nucleophilic amino acid in the active site of the enzyme. Serine proteases include many subfamilies.

[0152] In some embodiments, the protease comprises tryptase, the most abundant secretory granule-derived serine proteinase in mast cells and has been used as a marker for mast cell activation, released from mask cells when activated as part of normal immune and allergic responses.

[0153] In some embodiments, the protease comprises trypsin, which is a serine protease from the PA clan superfamily found in the digestive system. Trypsin cleaves peptide chains primarily at the carboxyl side of the amino acids lysine or arginine.

[0154] In some embodiments, the protease comprises urokinase (PLAU, uPA). Urokinase is a serine protease present in humans and other animals. It is present in human urine, blood, and the extracellular matrix of many tissues. It is involved in the degradation of the extracellular matrix and possibly tumor cell migration and proliferation. Urokinase is a 411-residue protein consisting of three domains: a serine protease domain, a kringle domain, and an EGF-like domain. Urokinase is synthesized as a zymogen form (pro-urokinase or single-chain urokinase) and is activated by proteolytic cleavage between Lys158 and Ile159. The two resulting chains are held together by disulfide bonds.

[0155] Agents to be detected are described herein, including, but not limited to, oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, proteases, hydrolases, esterases, β-glycosidases, phospholipases and phosphodiesterases, peroxidases, lipases, amylases, nucleophiles, reducing reagents, electrophilic / acidic reagents, organometallic / metal catalysts, oxidizing reagents, hydroxyl ions, thiol nucleophiles, nitrogen nucleophiles, sodium dithionite, and sodium periodate.

[0156] As described herein, the activity detection of some agents does not depend on cleavage. For example, some oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases result in substrate linker modification and the release or formation of a reporter molecule that can be detected. As an example, certain oxidation processes can modify inactive fluorophores and make them fluorescent / detectable without the need for a substrate linker, or binding events (in the case of non-covalent processes) can change the magnetic / fluorescent physicochemical properties of certain reporters and make them detectable.

[0157] Diseases and Conditions The method described herein comprises determining the disease or condition of the subject.In some aspects, the disease or condition comprises liver disease, cancer, metabolic disease, fibrotic disease, organ transplant rejection, infectious disease, allergic disease, autoimmunity, Alzheimer's disease, or chronic inflammation.In some embodiments, the liver disease comprises non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), toxin-mediated liver damage (drug / medicine, alcohol, environment), viral hepatitis (HAV, HBV, HCV, HDV, HEV, other viruses that infect the liver), autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, fulminant hepatitis, cirrhosis of the liver, hepatocellular carcinoma (HCC), cholangiocarcinoma, acute or chronic rejection of transplanted liver, hereditary liver disease (e.g., Wilson's disease, hemochromatosis, or alpha-1 antitrypsin), or combinations thereof.

[0158] In some embodiments, the cancer is adenoid cystic carcinoma, adrenal tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer, cholangiocarcinoma, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (bone sarcoma), brain stem glioma, brain tumor, breast cancer, Carney complex, central nervous system tumor, cervical cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, ependymoma, esophageal cancer, Ewing's sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, Familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, head and neck cancer, breast and ovarian cancer, diffuse gastric cancer, leiomyosarcoma and renal cell carcinoma, mixed polyposis syndrome, papillary renal carcinoma, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumors, laryngeal and hypopharyngeal cancer, leukemia, myeloid leukemia, lymphoblastic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung Cancer, Hodgkin's lung cancer, Non-Hodgkin's lung cancer, Lynch syndrome, Mastocytosis, Medulloblastoma, Melanoma, Meningioma, Mesothelioma, Multiple endocrine neoplasia, Multiple myeloma, Myelodysplastic syndrome, Nasal cavity and paranasal sinus cancer, Nasopharyngeal cancer, Neuroblastoma, Neuroendocrine tumors, Neurofibromatosis, Nevoid basal cell carcinoma syndrome, Oral cavity and oropharyngeal cancer, Osteosarcoma, Ovarian cancer, Fallopian tube cancer, Peritoneal cancer, Pancreatic cancer, Parathyroid cancer, Penile cancer, Peutz-Jeghers syndrome, Pheochromocytoma, including paraganglioma, pituitary tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, soft tissue sarcoma, sarcoma, non-melanoma skin cancer, small intestine cancer, gastric cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom's macroglobulinemia, Werner's syndrome, Wilms' tumor, or xeroderma pigmentosum.

[0159] In some embodiments, the disease includes hepatocellular carcinoma. Hepatocellular carcinoma, also called HCC or liver cancer, occurs when a tumor grows in or on the liver. HCC is the most common type of primary liver cancer and most commonly occurs in people with liver disease, especially those with chronic hepatitis B or C. HCC is also more common in people who drink large amounts of alcohol or have fat accumulation in the liver. Symptoms of HCC can include weight loss, upper abdominal pain, or yellowing of the skin (jaundice), although symptoms often do not appear early in the cancer. In some embodiments, the methods described herein can distinguish subjects with HCC from healthy subjects. In some embodiments, the methods described herein can distinguish subjects with HCC from subjects with only cirrhosis of the liver that has not developed into HCC.

[0160] In some embodiments, the disease includes a particular stage of HCC. No staging system for HCC is universally adopted. One system is the American Joint Committee on Cancer (AJCC) Tumor / Node / Metastasis (TNM) staging system. The TNM staging system takes into account tumor characteristics including size, number, and vascular invasion, as well as lymph node involvement and metastatic disease (Table 3). [Table 3]

[0161] In some embodiments, HCC detection using protease panel can be combined with additional markers of HCC, such as alpha-fetoprotein (AFP). AFP, also known as alpha-fetoglobin, is the major plasma protein produced by fetuses and is considered to be the fetal analog of serum albumin. A sustained increase in AFP serum levels can be a diagnostic marker for HCC and other chronic liver conditions.

[0162] Detection sensitivity and specificity The methods described herein include a detection sensitivity (also referred to herein as "sensitivity"). In some cases, the detection sensitivity of the method for HCC can be calculated by the following formula:

[0163] Sensitivity = number of samples with HCC and identified as positive / (number of samples with HCC and identified as positive + number of samples with HCC and identified as negative) or Sensitivity = number of samples with HCC and identified as positive / total number of samples with HCC.

[0164] In some embodiments, the detection sensitivity is at least about 0.5. In some embodiments, the detection sensitivity is at least about 0.51. In some embodiments, the detection sensitivity is at least about 0.52. In some embodiments, the detection sensitivity is at least about 0.53. In some embodiments, the detection sensitivity is at least about 0.54. In some embodiments, the detection sensitivity is at least about 0.55. In some embodiments, the detection sensitivity is at least about 0.56. In some embodiments, the detection sensitivity is at least about 0.57. In some embodiments, the detection sensitivity is at least about 0.58. In some embodiments, the detection sensitivity is at least about 0.59. In some embodiments, the detection sensitivity is at least about 0.6. In some embodiments, the detection sensitivity is at least about 0.61. In some embodiments, the detection sensitivity is at least about 0.62. In some embodiments, the detection sensitivity is at least about 0.63. In some embodiments, the detection sensitivity is at least about 0.64. In some embodiments, the detection sensitivity is at least about 0.65. In some embodiments, the detection sensitivity is at least about 0.66. In some embodiments, the detection sensitivity is at least about 0.67. In some embodiments, the detection sensitivity is at least about 0.68. In some embodiments, the detection sensitivity is at least about 0.69. In some embodiments, the detection sensitivity is at least about 0.7. In some embodiments, the detection sensitivity is at least about 0.71. In some embodiments, the detection sensitivity is at least about 0.72. In some embodiments, the detection sensitivity is at least about 0.73. In some embodiments, the detection sensitivity is at least about 0.74. In some embodiments, the detection sensitivity is at least about 0.75. In some embodiments, the detection sensitivity is at least about 0.76. In some embodiments, the detection sensitivity is at least about 0.77. In some embodiments, the detection sensitivity is at least about 0.78.In some embodiments, the detection sensitivity is at least about 0.79. In some embodiments, the detection sensitivity is at least about 0.8. In some embodiments, the detection sensitivity is at least about 0.81. In some embodiments, the detection sensitivity is at least about 0.82. In some embodiments, the detection sensitivity is at least about 0.83. In some embodiments, the detection sensitivity is at least about 0.84. In some embodiments, the detection sensitivity is at least about 0.85. In some embodiments, the detection sensitivity is at least about 0.86. In some embodiments, the detection sensitivity is at least about 0.87. In some embodiments, the detection sensitivity is at least about 0.88. In some embodiments, the detection sensitivity is at least about 0.89. In some embodiments, the detection sensitivity is at least about 0.9. In some embodiments, the detection sensitivity is at least about 0.91. In some embodiments, the detection sensitivity is at least about 0.92. In some embodiments, the detection sensitivity is at least about 0.93. In some embodiments, the detection sensitivity is at least about 0.94. In some embodiments, the detection sensitivity is at least about 0.95. In some embodiments, the detection sensitivity is at least about 0.96. In some embodiments, the detection sensitivity is at least about 0.97. In some embodiments, the detection sensitivity is at least about 0.98. In some embodiments, the detection sensitivity is at least about 0.99. In some embodiments, the detection sensitivity is at least about 1.

[0165] The methods described herein may have a detection specificity. In some cases, the detection specificity of the method for HCC may be calculated by the following formula:

[0166] Specificity = number of samples identified as not having HCC and negative / (number of samples identified as not having HCC and positive + number of samples identified as not having HCC and negative) or specificity = number of samples identified as not having HCC and positive / total number of samples not having HCC.

[0167] In some embodiments, the detection specificity is at least about 0.5. In some embodiments, the detection specificity is at least about 0.51. In some embodiments, the detection specificity is at least about 0.52. In some embodiments, the detection specificity is at least about 0.53. In some embodiments, the detection specificity is at least about 0.54. In some embodiments, the detection specificity is at least about 0.55. In some embodiments, the detection specificity is at least about 0.56. In some embodiments, the detection specificity is at least about 0.57. In some embodiments, the detection specificity is at least about 0.58. In some embodiments, the detection specificity is at least about 0.59. In some embodiments, the detection specificity is at least about 0.6. In some embodiments, the detection specificity is at least about 0.61. In some embodiments, the detection specificity is at least about 0.62. In some embodiments, the detection specificity is at least about 0.63. In some embodiments, the detection specificity is at least about 0.64. In some embodiments, the detection specificity is at least about 0.65. In some embodiments, the detection specificity is at least about 0.66. In some embodiments, the detection specificity is at least about 0.67. In some embodiments, the detection specificity is at least about 0.68. In some embodiments, the detection specificity is at least about 0.69. In some embodiments, the detection specificity is at least about 0.7. In some embodiments, the detection specificity is at least about 0.71. In some embodiments, the detection specificity is at least about 0.72. In some embodiments, the detection specificity is at least about 0.73. In some embodiments, the detection specificity is at least about 0.74. In some embodiments, the detection specificity is at least about 0.75. In some embodiments, the detection specificity is at least about 0.76. In some embodiments, the detection specificity is at least about 0.77.In some embodiments, the detection specificity is at least about 0.78. In some embodiments, the detection specificity is at least about 0.79. In some embodiments, the detection specificity is at least about 0.8. In some embodiments, the detection specificity is at least about 0.81. In some embodiments, the detection specificity is at least about 0.82. In some embodiments, the detection specificity is at least about 0.83. In some embodiments, the detection specificity is at least about 0.84. In some embodiments, the detection specificity is at least about 0.85. In some embodiments, the detection specificity is at least about 0.86. In some embodiments, the detection specificity is at least about 0.87. In some embodiments, the detection specificity is at least about 0.88. In some embodiments, the detection specificity is at least about 0.89. In some embodiments, the detection specificity is at least about 0.9. In some embodiments, the detection specificity is at least about 0.91. In some embodiments, the detection specificity is at least about 0.92. In some embodiments, the detection specificity is at least about 0.93. In some embodiments, the detection specificity is at least about 0.94. In some embodiments, the detection specificity is at least about 0.95. In some embodiments, the detection specificity is at least about 0.96. In some embodiments, the detection specificity is at least about 0.97. In some embodiments, the detection specificity is at least about 0.98. In some embodiments, the detection specificity is at least about 0.99. In some embodiments, the detection specificity is at least about 1. EXAMPLES

[0168] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein. It will be understood that variations and substitutions in the proportions of the components shown will be apparent to those skilled in the art and are within the scope of the embodiments presented herein.

[0169] Example 1. Diagnosis of HCC using the probes in humans In this experiment, the probes of the present application were shown to accurately detect the activity levels of hepatocellular carcinoma (HCC)-associated proteases in fluid samples to diagnose HCC in subjects.

[0170] In this experiment, we screened over 600 peptide substrates to identify a panel of 21 biosensors that interrogate multiple biological pathways involved in HCC pathogenesis. This panel of biosensors was highly effective in detecting HCC, with an AUC (area under the receiver operating characteristic curve) of over 0.94 when independently tested in two diverse cohorts of patients with known HCC versus healthy controls. In this study, the panel size was increased to 30 by adding 9 biosensors selected for their differential activity in mouse models of HCC versus fibrosis or fibrosis versus healthy.

[0171] Retrospective plasma samples were obtained from patients diagnosed with HCC or cirrhosis without HCC. Protease biosensor cleavage was assayed from plasma by fluorimetry and relative signals were used for classification by regularized logistic regression using 100-fold cross-validation (80% training, 20% validation split).

[0172] Protease activity levels associated with HCC were evaluated in vitro in two groups of human samples, healthy and those with virus-induced HCC. Table 4 shows the demographic data of the samples, and the results in Figure 5 show that protease activity significantly differentiates HCC vs. healthy samples. The experiment was repeated with lifestyle-induced HCC samples. Table 5 shows the demographic breakdown of the samples, and Figure 6 shows that protease activity similarly significantly differentiates HCC vs. healthy samples. [Table 4] [Table 5]

[0173] Figure 7 shows that plasma protease activity in 43 patients diagnosed with HCC (65% male, mean age 58 years, 40% Caucasian / 60% Asian, mean BMI 24 kg / m2, X etiology, 72% early stage I or II) was compared with 26 patients with underlying cirrhosis only (30% male, mean age 57 years, 100% Caucasian, mean BMI 36 kg / m2, 100% NASH etiology). The protease biosensor was effective in distinguishing HCC from cirrhosis (AUC=0.93 [CI 0.86-0.98]). Furthermore, using the best cut point under the AUROC, the sensitivity and specificity were 0.84 and 0.85, respectively. The most important biosensors in HCC vs. cirrhosis classification are MMPs, DPP4, ANPEP, aminopeptidases (ERAP1, ARTS-1), KLK14, and cathepsins (L, S, K, B), as well as substrates of ADAM17. HCC signals generalize across cohorts without classifier retention (Figure 8).

[0174] Thus, the probe of the present application can accurately detect protease activity levels associated with biological or disease states in subjects ex vivo using bodily fluid samples. A novel non-invasive biosensor measuring protease activity can distinguish HCC (AUC 0.93) compared to background cirrhosis alone. Consistent with other data in HCC vs. healthy and NASH vs. healthy, measuring protease activity is a potentially important new diagnostic platform.

[0175] Example 2: Detection of HCC stage using protease activity As shown in Figures 9A-9B, protease activity can not only detect differences between healthy and diseased samples, but also distinguish between stages of HCC in both virus-induced (Figure 9A) and lifestyle-induced (Figure 9B) HC samples. This experiment used the same method as described in Example 1.

[0176] Example 3: Plasma protease activity and alpha-feto-protein (AFP) for detecting HCC HCC surveillance using ultrasound can be combined with detection of alpha-feto-protein (AFP). In this study, AFP detection was combined with a protease biosensor biomarker panel to evaluate the efficacy of the combined treatment to distinguish patients with HCC from those with cirrhosis without HCC.

[0177] Retrospective plasma samples were obtained from patients diagnosed with HCC (cases) and patients with cirrhosis without HCC (controls). Protease biosensor cleavage was assayed from plasma by fluorimetry. Relative signals were used for classification by regularized logistic regression using 100 cross-validation (80% training, 20% validation split). The maximum point from Youden's J index in the training set was used to define the optimal cutoff, which was used to calculate the sensitivity and specificity of HCC detection in the validation set. AFP was measured in plasma samples of all patients using a standard ELISA assay. In this analysis, AFP thresholds at both 20 ng / mL and 10 ng / mL were evaluated.

[0178] Plasma protease activity and AFP levels were evaluated in 54 HCC cases (76% male, mean age 56 years, HBV 65%, TNM stage 1 or 2 82%) and 23 cirrhotic controls (35% male, mean age 58 years, 100% NASH etiology). Our protease biosensor panel had high accuracy for discriminating patients with HCC versus those with cirrhosis (AUC 0.87 [95% CI 0.78-0.94]). The sensitivity and specificity of the assay were 0.79 and 0.77, respectively, at its prespecified cutoff. The sensitivity and specificity of AFP at a cutoff of 20 ng / mL were 0.44 and 1.00, respectively. Of note, the assay was able to detect HCC in 68% (22 of 30) of patients with AFP < 20 ng / mL. Finally, the combined classifier of the assay and AFP > 20 ng / mL had an AUC of 0.90 [95% CI 0.82-0.96] for distinguishing HCC from cirrhosis. The sensitivity and specificity of the combined classifier for HCC detection were 0.82 and 0.82, respectively. Similar results were observed when the AFP threshold was adjusted to a more stringent cutoff of 10 ng / mL.

[0179] As seen in Figure 10A-B, the protease biosensor panel assay was able to correctly classify as HCC 68% (17 of 25) of patients with low AFP levels (AFP < 10 ng / ml). Furthermore, classification by regularized logistic regression with 100-fold cross-validation (80% training, 20% validation split) using the protease biosensor panel assay in the challenging subset of patients with low AFP levels (AFP, 10 ng / ml) showed an AUC of 0.75. Finally, the combined classifier of the protease biosensor panel assay and AFP had an AUC of 0.90 [CI 0.83-0.96] for distinguishing HCC from cirrhosis in this cohort of patients (Figure 10B). Using Youden's cutoff, the sensitivity and specificity for HCC detection of this combined classifier of the protease biosensor panel + AFP were 0.84 and 0.87, respectively. These combined results demonstrate the utility of using a novel noninvasive biosensor that measures protease activity, either independently or in combination with AFP, to accurately distinguish HCC from cirrhosis.

[0180] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in the practice of the present invention. Thus, the present invention is also intended to cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims, and their equivalents, are covered thereby.

Claims

1. 1. A method comprising: contacting a bodily fluid sample from a subject with a synthetic molecule, said contacting occurring ex vivo; the subject has or is suspected of having cirrhosis of the liver of the subject; the synthetic molecule comprises a cleavable linker, a reporter, and a carrier; said contacting, wherein said synthetic molecule reacts with an enzyme from said bodily fluid to cause said reporter to form a detectable signal; detecting the detectable signal, and detecting, wherein the detecting indicates whether the subject has hepatocellular carcinoma (HCC), and the detecting comprises a detection sensitivity of at least 0.

5.

2. The method of claim 1 , wherein the detection sensitivity is at least 0.

8.

3. 2. The method of claim 1, wherein the detection sensitivity is calculated by dividing the number of samples identified as having HCC and being positive by the total number of samples, the total number of samples including the sum of the samples identified as having HCC and being positive and the samples identified as having HCC and being negative.

4. The method of claim 1 , wherein the HCC comprises a stage of HCC.

5. The method of claim 1, wherein the enzyme from the bodily fluid cleaves the cleavable linker, thereby releasing the reporter from the synthetic molecule, and the released reporter forms the detectable signal.

6. The method of claim 1 , wherein the cleavable linker comprises a peptide.

7. The method of claim 1 , wherein the synthetic molecule comprises a panel of peptides.

8. The method of claim 7 , wherein the panel of peptides comprises at least 10 different peptides.

9. The method of claim 1 , wherein the enzyme comprises a protease.

10. 10. The method of claim 9, wherein the protease comprises a matrix metalloprotease (MMP), dipeptidyl-peptidase 4 (DPP4), alanyl aminopeptidase (ANPEP), aminopeptidase (ERAP1, ARTS-1), kallikrein-related peptidase 14 (KLK14), cathepsin, or ADAM17.

11. The method of claim 1 , wherein the reporter comprises a fluorescent label.

12. 12. The method of claim 11, wherein the fluorescent label comprises 5-carboxyfluorescein (5-FAM), 7-amino-4-carbamoylmethylcoumarin (ACC), 7-amino-4-methylcoumarin (AMC), 2-aminobenzoyl (ABZ), Cy7, Cy5, Cy3, or (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid) (EDANS).

13. The method of claim 1 , wherein the synthetic molecule further comprises a fluorescence quencher.

14. 14. The method of claim 13, wherein the fluorescence quencher comprises BHQ0, BHQ1, BHQ2, BHQ3, BBQ650, ATTO 540Q, ATTO 580Q, ATTO 612Q, CPQ2, QSY-21, QSY-35, QSY-7, QSY-9, DABCYL (4-([4'-dimethylamino)phenyl]azo)benzoyl), Dnp (2,4-dinitrophenyl), or Eclipse.

15. 2. The method of claim 1, wherein the bodily fluid sample comprises blood, plasma, bone marrow fluid, lymphatic fluid, bile, amniotic fluid, mucosal fluid, saliva, urine, cerebrospinal fluid, spinal fluid, synovial fluid, semen, ductal aspirate, feces, stool, vaginal flow, tears, tissue lysate, or patient-derived cell line supernatant.

16. 10. The method of claim 1, wherein the bodily fluid sample comprises a rinse fluid, a conditioning medium or buffer, a swab virus transport medium, saline, a culture medium, or a cell culture supernatant.

17. 17. The method of claim 16, wherein the rinse comprises a mouthwash rinse, a bronchoalveolar rinse, a lavage fluid, a hairwash rinse, a nasal spray effluent, a swab of any body surface, orifice, organ structure, or solid tumor biopsy applied to saline or any medium, or any derivative thereof.

18. 10. The method of claim 1, wherein the bodily fluid sample comprises an alpha-fetoprotein (AFP) level of less than about 10 ng / ml.

19. The method of claim 1 or 10, wherein said detecting further comprises detecting an AFP level in said body fluid sample.

20. The method of claim 1 , wherein the detecting comprises detecting the rate or amount of formation of the detectable signal.

21. The method of claim 1 , wherein the subject comprises a mammal.

22. The method of claim 1 , wherein the subject comprises a human.

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