Compositions and methods for logic-gated profiling of biological activity

Abstract

The present disclosure provides methods and compositions for digitally profiling biological activity by detecting the activity of at least two biomarkers using a protease activity sensor or biocomparator.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 811,619, filed February 28, 2019, which is incorporated by reference herein in its entirety. Government support explained This invention was made with government support under Grant No. DP2HD091793 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0002] Technical Field The present disclosure relates to tools, compositions, and methods for digitally profiling biological activity. [Background technology]

[0003] Advances in human health are premised on an increasing ability to extract meaningful information from complex biological states and use this information to guide diagnosis, prognosis, or therapeutic intervention. Currently, clinical decisions rely on obtaining information from complex biological states by examining "analog" signals, such as biomarker levels, which are prone to noise, imprecision, and heterogeneity. Applying principles from computing to biological systems can improve the accuracy and sensitivity of obtaining information from complex biological states, leading to accurate disease diagnosis and improved medical treatment outcomes. For example, computing uses "digital" signals that robustly convey information and are resistant to uncontrolled variables. The use of compositions and methods that enable profiling biological activity under a digital framework, such as Boolean logic, can dramatically improve our ability to extract meaningful information from complex biological states.

[0004] The enzymatic activity of specific proteases has been shown to be of paramount importance in the monitoring and diagnosis of specific disease states and treatments. Over 550 proteases are encoded by the human genome, and dysregulation of protease signaling networks underlies the biology of numerous disease states, including cancer, fibrosis, hematological disorders, and immune disorders (Mason and Joyce, Trends Cell Biol., 21(4):228-237 (2011)). Thus, detection of protease activity can provide meaningful information from complex biological states and prove useful for diagnosis, prognosis, and the development of therapeutic treatments. This is particularly true for cancer, where proteases play a key role in inducing tumorigenesis.

[0005] Accurate detection of protease activity can also provide information about the efficacy and specificity of specific therapeutic treatments. An important and emerging class of therapeutic treatment is immunotherapy, which harnesses the immune system to treat a myriad of diseases, such as cancer, organ transplant rejection, infectious diseases, allergic diseases, autoimmunity, and chronic inflammation. These treatments employ both the humoral and cellular arms of the immune response, using therapeutic antibodies, cytokines, and cell-based therapies. Despite the broad potential of immunotherapy, many patients fail to experience clinical benefit, and others may develop resistance to immunotherapy. Patients who respond to immunotherapy often exhibit unconventional response patterns that can be mistaken for disease progression. Due to inadequate technologies for monitoring response and identifying underlying resistance mechanisms, not only does the disease persist in the population, but drug development and clinical trials face significant obstacles. To realize the full benefits of immunotherapy, improved methods for monitoring biomarkers during immunotherapy, including protease activity, are needed.

[0006] Traditional methods for measuring protease activity, such as structural biology, enzymology, and inhibitor-based assays, have provided important information about proteases. However, these techniques often lack sensitivity and cannot distinguish between different disease states or off-target activity.

[0007] In the context of diagnosis, tissue biopsy remains the gold standard, but it is invasive, and samples are obtained from less than 0.1% of all disease sites (Cyll et al., Br J Cancer, 117(3):367-375 (2017)). Liquid biopsies offer a noninvasive approach, but sensitivity is significantly limited by biomarker dilution in the blood (Nagrath, S., et al., Nature, 450(7173): 1235-1239 (2007); Hori, et al., Sci Transl Med, 3(109): l09ral6 (2011)). Imaging techniques can also be limited by low sensitivity and specificity, as well as by nontraditional response patterns commonly associated with immunotherapy, which can lead to misidentification of responders, such as in cases of treatment failure. Developing better, noninvasive biomarkers will identify responders sooner and elucidate the mechanisms of novel immunotherapies. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Mason and Joyce, Trends Cell Biol., 21(4):228-237 (2011) [Non-patent document 2] Cyll et al., Br J Cancer, 117(3):367-375 (2017) [Non-patent document 3] Nagrath, S., et al, Nature, 450(7173): 1235-1239 (2007) [Non-patent document 4] Hori, et al., Sci Transl Med, 3(109): l09ral6 (2011) Summary of the Invention [Problem to be solved by the invention]

[0009] It is therefore an object of the present invention to provide biological activity sensors that exploit protease biology for specific, sensitive, and non-invasive diagnostics. [Means for solving the problem]

[0010] The present disclosure provides methods and compositions for digitally profiling biological activity by detecting the activity of at least two biomarkers. The biomarkers can be proteins or enzymes, such as proteases and / or cytolytic proteins. The methods and compositions detect biomarker activity using a digital framework. To establish the digital framework, the present disclosure provides a logic-gated biomarker activity sensor. The activity sensor probes the activity of two biomarkers using AND-gated logic. The activity sensor senses biological activity by a specific biomarker as a bit. Activity of one biomarker represents a "1" state, and lack of activity represents a "0" state. Activity of two biomarkers represents a "1,1" state, providing a TRUE output. Activity of only one of the two biomarkers represents a "1,0" state, and lack of biomarker activity represents a "0,0" state. Both the "0,1" and "0,0" states provide a FALSE output.

[0011] An activity sensor can include a protease substrate designed to sense biological activity as a biological bit through a specific protease cleavage event, with an uncleaved substrate representing a "0" state and a cleaved substrate representing a "1" state. The sensor simultaneously probes the activity of two proteases by using AND-gated logic to provide a TRUE output only after cleavage events by both proteases. Cleavage of one or neither of the protease substrates results in a FALSE output. Using this concept, the present disclosure provides methods and compositions using logic-gated activity sensors that exploit protease activity to provide, for example, specific, sensitive, and non-invasive diagnostics.

[0012] The present disclosure provides methods and compositions for determining protease activity in a biological sample using a protease activity sensor. The protease activity sensor includes a first protease substrate and a second protease substrate. Each substrate is cleaved by a different protease, e.g., the first protease and the second protease. The sensor can employ AND-gated logic. Thus, if both substrates are cleaved, the protease activity sensor provides a detectable signal (TRUE output), indicating the activity of both the first and second proteases in the sample. However, if only one or neither of the substrates is cleaved, no detectable sample is generated (FALSE output).

[0013] To provide a detectable signal, the protease sensor can be conjugated to a reporter molecule, such as a fluorescent molecule. If the reporter molecule is a fluorescent molecule, the first and second protease substrates can be conjugated to a fluorescent quencher. Cleavage of the protease substrate removes the quencher, allowing the fluorescent molecule to provide a detectable signal (TRUE output). However, if one or both protease substrates are not cleaved, the fluorescent quencher is not removed, preventing the detectable signal (FALSE output).

[0014] The protease sensor can be a cyclic peptide. The cyclic peptide can include two separate protease substrates. The substrates can separate the fluorescent reporting molecule and the quencher.

[0015] The protease sensor can be conjugated to a scaffold such as a nanoparticle. Multiple protease sensors can be conjugated to a scaffold. Conjugating the protease sensor to a scaffold increases the valency of presentation, increasing the rate of proteolysis and thus amplifying the detectable signal. This can improve the signal-to-noise ratio, especially when the protease sensor is delivered in vivo.

[0016] The present disclosure provides methods and compositions for determining biomarker activity in a biological sample using a bicomparator. The bicomparator can include a reporting molecule encapsulated in a liposome. The liposome is contained within a peptide cage. Cleavage of the peptide cage by a protease releases the liposome. The liposome can be perforated by a cytolytic protein, such as perforin, potentially releasing the reporter molecule. The bicomparator can employ AND-gated logic. Upon release, the reporter molecule provides a detectable signal (TRUE output), indicating the activity of both the protease and the cytolytic protein in the sample. In the absence of a protease capable of cleaving the peptide cage and the cytolytic protein to perforate the liposome, the reporter molecule is not released and no detectable signal is generated (FALSE output). The reporter molecule can be a fluorescent molecule.

[0017] Bicomparator can be conjugated to a scaffold such as nanoparticles.Multiple biocomparators can be conjugated to a scaffold.Conjugating multiple bicomparators to a scaffold increases the display valency and increases the rate of proteolysis, thereby amplifying the detectable signal.This can improve the signal-to-noise ratio, especially when the bicomparator is delivered in vivo.

[0018] The protease substrate, including the bicomparator peptide cage, is designed to be cleaved by a specific protease. This allows the presence of a specific protease in a sample to be determined. The presence or absence of a specific protease can indicate protease dysregulation. Protease dysregulation can indicate the presence of a disease state such as cancer, fibrosis, blood disease, immune disease, viral infection, or bacterial infection. Therefore, the disclosed methods and compositions can be used to diagnose disease or disease progression.

[0019] Protease activity can indicate an immune response to immunotherapy or therapeutic drugs. Therefore, the disclosed methods and compositions can be used to monitor immunotherapy and therapeutic drug treatment. Monitoring can include measuring the efficacy, specificity, and response of a particular treatment. This capability of the disclosed methods and compositions is particularly useful for cancer immunotherapy.

[0020] Monitoring the immune response can involve detecting the activity of one or more proteases and / or cytolytic proteins. The activity of one or more proteases and / or cytolytic proteins can be stimulated by a therapeutic agent. In this manner, the activity, specificity, and / or efficacy of the therapeutic agent can be monitored.

[0021] Cancer immunotherapy is limited by its off-target effects. A method for non-invasively detecting intratumoral immune activity would substantially improve treatment monitoring. Simply monitoring a patient's immune activity cannot confirm the effectiveness of immunotherapy, for example, because the immune system is activated against other pathologies (e.g., viral infection). The specificity provided by the AND-gated logic protease activity sensor and bicomparator makes it possible, for example, to distinguish immune activity in tumors from immune activity caused by viral infection. The methods and compositions disclosed herein enable monitoring of intratumoral immune activity and can distinguish between immune activity caused by tumors, therapeutic treatments, and other pathologies such as viral infection.

[0022] The disclosed methods and compositions can be used in vitro or in vivo. The disclosed methods can include administering to a subject an amount of a protease sensor or bicomparator described herein. The methods can include monitoring the activity of one or more proteases and / or cytolytic proteins in the subject. The activity of the proteases and / or cytolytic proteins can indicate protease dysregulation and / or an immune response in the subject. The protease dysregulation can indicate the presence of a disease state in the subject. The disease state can be cancer, fibrosis, a blood disorder, an immune disorder, a viral infection, or a bacterial infection. The activity of the proteases and / or cytolytic proteins can be promoted by a therapeutic agent. The methods can include monitoring the activity of at least one of granzyme B (GzmB), thrombin (Thrb), metalloproteinase (MMP), or viral protease. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 illustrates the principle behind digital readout of biological activity. [Figure 2]FIG. 2 shows a schematic diagram of an example of a protease activity sensor. [Figure 3] FIG. 3 shows the principle behind the AND gating logic in the protease activity sensor. [Figure 4] Figure 4 shows a schematic diagram of an AND-gated biocomparator. [Figure 5] Figure 5 shows a schematic diagram of a biocomparator in which the peptide cage is cleaved. [Figure 6] FIG. 6 shows a schematic diagram of the biocomparator in which the liposomes are perforated and the reporter molecules are released. [Figure 7] FIG. 7 illustrates a representative method of the present disclosure. [Figure 8] FIG. 8 shows a representative cyclic protein activity sensor. [Figure 9] FIG. 9 shows a schematic diagram of a representative protease activity sensor. [Figure 10] FIG. 10 shows experimental results for a representative protease activity sensor. [Figure 11] FIG. 11 shows the experimental results for a representative protease activity sensor. [Figure 12] FIG. 12 shows the experimental results for a representative protease activity sensor. [Figure 13] FIG. 13 shows experimental results for a representative protease activity sensor. [Figure 14] FIG. 14 shows experimental results for a representative protease activity sensor. [Figure 15] FIG. 15 shows a schematic diagram of a protease activity sensor. [Figure 16] FIG. 16 shows the experimental results for the protease activity sensor. [Figure 17] FIG. 17 shows the experimental results for the protease activity sensor. [Figure 18] FIG. 18 shows a schematic diagram of a protease activity sensor. [Figure 19]FIG. 19 shows the experimental results for the protease activity sensor. [Figure 20] FIG. 20 shows the experimental results for the protease activity sensor. [Figure 21] FIG. 21 shows the experimental results for the protease activity sensor. [Figure 22] FIG. 22 shows the experimental results for the biocomparator. [Figure 23] FIG. 23 shows the experimental results for the biocomparator. [Figure 24] FIG. 24 shows a schematic diagram of the experimental protocol using the biocomparator. [Figure 25] FIG. 25 shows a schematic diagram of the biocomparator and the experimental results. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present disclosure provides methods and compositions for digitally profiling biological activity by detecting the activity of at least two biomarkers. Biomarkers can include proteins or enzymes, such as proteases and / or cytolytic proteins. The methods and compositions detect biomarker activity using a digital framework. To establish the digital framework, the present disclosure provides a logic-gated biomarker activity sensor. The methods and compositions use AND-gated logic to probe the activity of two biomarkers. The methods and compositions sense biological activity due to a specific biomarker as a bit. Activity of one biomarker represents a "1" state, and lack of activity represents a "0" state. Activity of two biomarkers represents a "1,1" state, providing a TRUE output. Activity of only one of the two biomarkers represents a "1,0" state, and lack of biomarker activity represents a "0,0" state. Both the "0,1" and "0,0" states provide FALSE outputs. Because the methods and compositions require the activity of two specific biomarkers, they can reliably provide a digital profile of biological activity.

[0025] Enzymes are differentially expressed under various physiological conditions of interest, such as in response to disease, infection, immune response, or therapeutic treatment. For example, dysregulated protease activity can indicate a disease state. Dysregulated proteases have important consequences for disease progression, including cancer, in that they can alter cell signaling and promote cancer proliferation, invasion, angiogenesis, apoptosis evasion, and metastasis. However, the activity of a single enzyme, including a protease, is often the determining factor for a biological response. By detecting the activity of multiple enzymes, such as proteases and cytolytic proteins, responses can be more reliably determined and reported using binary signals, such as TRUE or FALSE outputs.

[0026] The methods and compositions are useful for monitoring and diagnosing disease, immune response, and therapeutic activity. Monitoring can include, for example, long-term monitoring, including changes in disease state, immune activity, and / or response to therapeutic activity. The methods and compositions are useful for long-term monitoring and personalized medicine, as well as for recruiting, enriching, qualifying, and stratifying study participants. The methods and compositions are useful for detecting the effectiveness of treatment, monitoring response to treatment over time, and detecting relapse and remission. Detection of the reporter in a sample from a subject can indicate the presence of disease in the subject, the stage of disease, and the rate or level of disease activity. Tests using the compositions of the present disclosure can be administered and read non-invasively, quickly, and without imaging by X-ray or other modalities.

[0027] The compositions of the present disclosure can be used to determine a subject's responsiveness to a drug and the effectiveness of treatment. For example, the compositions can be administered and tested at multiple time points and read to observe trends over time in disease change or progression or remission. Regardless of whether the subject has received treatment or whether the composition is used diagnostically, the compositions can be used to measure activity in the body. Thus, the present invention provides a better understanding of disease, rather than simply its effects. The compositions can be used to study disease and / or other related activities. For example, disease activity or progression, such as tumor growth rate, can be detected. In the case of subjects receiving drug treatment, the compositions can be used to detect activity indicating whether the participant is responding to treatment. For example, activity sensors can be designed to detect whether tumor size is growing or shrinking, the rate of disease activity or progression, whether a particular drug dosage is effective, and whether the participant is likely to respond to treatment.

[0028] The method and composition can be used to detect the activity of two specific biomarkers to digitally profile whether a subject responds to multiple treatments. By measuring the activity of two specific biomarkers, the method and composition of the present disclosure allows the type of response in a subject to be more accurately determined, allowing the use of a binary signal. For example, a specific drug can be used to treat a disease. However, the drug may be intended for different methods of treating the disease. When treating a disease such as nonalcoholic steatohepatitis (NASH), one drug can be an anti-fibrotic drug, and the other drug can be an anti-inflammatory or anti-NASH drug. Detecting the activity of both drugs by examining the activity of biomarkers associated with each drug produced in the participant's body provides insight into why the participant responded or did not respond to the treatment.

[0029] Furthermore, the method and composition can be used to detect whether a subject is responding to treatment. For example, the subject can have a tumor that has been or is being treated with a checkpoint blockade. After administering checkpoint blockade, the tumor continues to grow in size, so the subject may appear to be unresponsive to treatment. However, because the present invention detects the biological activity of two biomarkers in the body, the present invention can detect that the growth is due to the influx of immune cells. At the same time, due to the specificity provided by the method and composition of the present disclosure, it can distinguish whether the immune response is caused by a checkpoint blockade or a pathology such as a viral infection. Therefore, it is determined that the subject is not unresponsive, but is responding to treatment.

[0030] The disclosed methods and compositions can include protease activity sensors. Such activity sensors can include various reporter molecules that are detectable in a sample but only upon contact and cleavage by two proteases associated with a local immune response or cancer progression. Cleavage by only one protease does not result in a state represented by "1,1," thus providing a FALSE output. Cleavage by both proteases results in a state represented by "1,1," thus providing a TRUE output. The protease sensor can be provided to a subject in vivo, and the reporter molecule can be detected in bodily fluids upon protease cleavage, which releases the reporter molecule.

[0031] As shown in Figure 1A, the protease activity sensor of the present disclosure allows for the provision of a digital readout of biological activity. As described herein, the protease activity sensor can provide non-invasive reporting of the activity of two proteases through the engineering of two protease-specific cleavage sites on the sensor. For example, the protease-specific substrates (e.g., cleavage sites) can be cleaved by proteases (one or more) promoted by cancer or immune activity.

[0032] FIG. 2 provides a non-limiting schematic diagram of a protease activity sensor. As shown in FIG. 2, multiple protease activity sensors can be attached to a scaffold, such as a nanoparticle. Each protease activity sensor has a reporter molecule, indicated by an asterisk in FIG. 2. The reporter molecule can be a fluorescent molecule. The reporter molecules are attached to two different protease substrates and are cleaved by the activity of two different proteases. As shown in FIG. 2, when the reporter molecule is a fluorescent molecule, the protease activity sensor can include a fluorescent quencher, indicated by a black circle in FIG. 2. The fluorescent quencher prevents the fluorescent molecule from providing a detectable signal. Upon cleavage by the two different proteases, the reporter molecule is released. As shown in FIG. 2, the release can allow the reporter molecule to move away from the fluorescent quencher, thereby enabling a detectable signal.

[0033] Figure 3 shows how the protease activity sensor provides AND-gated logic to simultaneously probe the activity of two proteases by providing a TRUE output only after a cleavage event by both proteases. A signal of "0,0" results from no protease cleavage and provides a FALSE output. A signal of "0,1" results from protease cleavage by only one protease and provides a FALSE output. A signal of "1,1" results from protease cleavage by both proteases and provides a TRUE output.

[0034] The disclosed methods and compositions can include a bicomparator. The bicomparator can include various reporter molecules. The reporter molecule is encapsulated in a liposome. The liposome is contained within a peptide cage that is a substrate for a specific protease(s). Cleavage of the peptide cage by the protease releases the liposome. The liposome can be perforated by a cytolytic protein, such as perforin. The reporting molecule is detected only upon cleavage of the peptide cage by the protease and perforation of the liposome by the cytolytic protein. Release of the reporter molecule results in a state represented by "1,1," thus providing a TRUE output. The bicomparator can be provided to a subject in vivo, and the reporter molecule becomes detectable in bodily fluids upon release of the reporter molecule.

[0035] Figures 4-6 provide non-limiting schematic diagrams of a bicomparator. As shown in Figure 4, bicomparator 401 has a reporter molecule 404, shown as a star. The reporter molecule may be a fluorescent molecule. Reporter molecule 404 is encapsulated within a liposome 403. Liposome 403 is contained within a peptide cage 402. A protease 405 contacts bicomparator 401. As shown in Figure 5, the protease cleaves the peptide cage. Then, as shown in Figure 6, a cytolytic protein 606, such as perforin, punctures the liposome, releasing the reporter molecule and thereby providing a detectable signal.

[0036] The present disclosure encompasses methods for administering a protease activity sensor or bicomparator of the present disclosure to a subject in vivo to monitor or diagnose disease or treatment effectiveness. FIG. 7 illustrates a non-limiting example of such a method 701. Method 701 can be performed in the context of a longitudinal study to determine the effectiveness of a therapeutic treatment. Subject 705 includes a subject who will receive or is receiving the treatment. A protease activity sensor or bicomparator is administered to the subject (705). Method 701 can encompass monitoring changes in disease state in a subject receiving therapeutic treatment for a disease by taking measurements over time (i.e., longitudinally) at multiple time points. Two specific biomarkers contact and interact with the protease activity sensor or bicomparator (707). This causes the protease activity sensor or bicomparator to release a reporter molecule 709. A sample is obtained from the subject (711) and an assay is performed to detect a signal in the sample (713). By detecting the reporter in the sample from the subject, the activity, and therefore the presence, of two biomarkers in the sample is identified. The presence and activity of the two biomarkers in the sample can indicate a progression in a disease state or immune response that is promoted by therapeutic treatment.

[0037] Method 701 can be used to detect disease-related activity in the body, such that the activity of two biomarkers detected using the sensors indicates disease progression or indicates that a treatment is effective. A protease activity sensor or bicomparator can be administered over time to monitor a subject's response to treatment and indicate whether the treatment is effectively treating the subject's disease. Different drugs or drug combinations can be studied in different subjects or at different time points to identify drugs or combinations that work well to treat a condition. A protease activity sensor or bicomparator provides a marker of health / disease progression, and does so very quickly (detection of a signal in a sample from a subject can occur within a few hours of administering the protease activity sensor or bicomparator). A protease activity sensor or bicomparator can specifically report the activity of multiple enzymes, including those whose expression is deregulated in disease states and others specific to co-morbidities. Preferably, the enzyme (eg, an extracellular protease) has expression that is upregulated in response to a particular lesion, treatment, or condition.

[0038] Method 701 may be used to monitor the progression of cancer in a subject. The subject may be suspected of having cancer, known to have cancer (active or in remission), at risk of developing cancer, and / or undergoing cancer treatment, including cancer immunotherapy (IO). A protease sensor may be administered to the subject (705). The sensor may include a reporter linked by two protease substrates. Each of the protease substrates is sensitive to a protease whose activity is characteristic of the tumor environment (e.g., an enzyme upregulated in expanding or regressing tumors, or an enzyme indicative of an active or inhibited immune response).

[0039] Similarly, a biocomparator can be administered to the subject (705). The biocomparator can have a peptide cage that is a substrate for a protease, the activity of which is characteristic of the tumor environment. The liposomes of the biocomparator can be perforated by a protein whose activity is characteristic of the tumor environment.

[0040] As discussed herein, the activity sensor can be operated to report on the patient's disease and treatment status depending on the protease activity, and information obtained from the reporter level in a patient sample can be used to diagnose and / or stage the disease, monitor progression, predict responsiveness to a given treatment, and monitor treatment effectiveness, including distinguishing between anti-tumor immune responses, general immune responses, and tumor progression. The activity sensor can be administered by any suitable method. The activity sensor can be delivered intravenously or aerosolized and delivered to the lungs, for example, via a nebulizer. In other examples, the activity sensor can be administered to a subject transdermally, intradermally, intra-arterially, intralesionally, intratumorally, intracranially, intra-articularly, intratumorally, intramuscularly, subcutaneously, orally, topically, locally, by inhalation, injection, infusion, or by any other method or combination known in the art.

[0041] Proteases are a class of enzymes with over 550 members encoded in the human genome, many of which have disease-specific roles, including important roles in immunity. For example, cytotoxic T cell-mediated target cell killing is a protease-driven process involving: 1) death receptor signaling and activation of caspases, proteases whose activity mediates cell death, and 2) secretion of granzymes, proteases that enter target cells via a perforin-dependent mechanism and activate caspase-mediated cell death. Furthermore, proteases are central to other aspects of immune activity, including cell migration, matrix degradation and repair, and complement activation, while tumor proteases, such as inflammatory and matrix-degrading proteases, have been established as hallmarks of cancer (Arias, et al., Trends Cancer, 3(6):407-422 (2017); Egeblad, et al., Nai Rev Cancer, 2(3):161-174 (2002)).

[0042] Proteases offer an innovative approach for immunotherapy response monitoring, given their central role in the biology underlying immunity, oncology, and the pathophysiology of multiple diseases (Dudani, et al., Ann Rev of Cancer Biology, (2018)). For example, the "inflammatory" tumor signature is marked by effective immune infiltration of cytotoxic T cells, which kill cancer cells primarily through a perforin-dependent granzyme-mediated pathway, and granzymes comprise a family of potent serine proteases (Larimer, et al., Cancer Res, 77(9):2318-2327 (2017); Voskoboinik, et al., Nat Rev Immunol, 15(6):388-400 (2015)). Tumor expression of proteases, including inflammatory and matrix-degrading proteases, is well established as a hallmark of fundamental tumor biology, including angiogenesis, growth, and metastasis (Dudani, et al., Ann Rev of Cancer Biology, (2018)). These protease signatures can be used to stage cancer, monitor progression and regression, and provide early indicators of drug response. Early immune and disease-site-specific protease activity during treatment allows for the identification of active biomarkers that predict treatment efficacy and indicate resistance to immunotherapy.

[0043] In one embodiment, the protease amplifies the detection signal (×1000 fold) at the site of disease or treatment. Following protease cleavage, the reporter molecule becomes concentrated in the urine instead of being diluted in the blood, further enhancing the signal by up to 100-fold. This allows, for example, ultrasensitive early detection of T cell activity prior to detectable radiological changes at the site of disease.

[0044] The protease substrate contains a recognition sequence for cleavage by a protease. Cleavage of the protease substrate can release a reporter molecule linked to the substrate. The protease substrates can each be a substrate for a particular protease known to be associated with diseased cells. Proteases known to be associated with diseased cells or tissues include, but are not limited to, serine proteases, cysteine ​​proteases, aspartic acid proteases, threonine proteases, glutamic acid proteases, metalloproteases, aspartic peptide lyases, serum proteases, cathepsin, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidain, bromelain, calpain, and caspases. , caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteinases (MMPs), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, meprin, granzyme, and dipeptidyl peptidase IV (DPPIV / CD26).

[0045] The protease substrate can be a tumor-specific protease substrate. Representative tumor-associated proteases include, but are not limited to, cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein 1, kallikrein 3 (PSA), kallikrein 10, kallikrein 5, uPA, uPAR, caspases, matrix metalloproteinases such as MMP1, MMP2, MMP8, MMP9, MMP13, MMP14, and ADAM. In other embodiments, the protease substrate is a cell-specific protease substrate, such as a T cell-specific protease substrate. Representative cell-specific proteases include, but are not limited to, neutrophil serine proteases such as cathepsin G, neutrophil elastase, and proteinase 3, mucosa-associated lymphoid tissue 1 (MALT1), granzymes, and cysteine ​​proteinases of the caspase family, e.g., caspase-3, -6, -7, -8.

[0046] The protease substrate can be a substrate specific to proteases associated with inflammation and / or programmed cell death. Several proteases are known to be associated with inflammation and programmed cell death (e.g., apoptosis, pyroptosis, and necroptosis). Therefore, the activity level of these proteases indicates immune system activity. Caspases (cysteine-aspartic acid proteases, cysteine ​​aspartases, or cysteine-dependent aspartate-directed proteases) are a family of protease enzymes that contain a cysteine ​​in their active site that nucleophilically cleaves target proteins only after aspartic acid residues. Caspase-1, caspase-4, caspase-5, and caspase-11 are associated with inflammation. Serine proteases also function in apoptosis and inflammation, and therefore, their differential expression also indicates immune response. Immune cells express serine proteases such as granzymes, neutrophil elastase, cathepsin G, proteinase 3, chymase, and tryptase.

[0047] The disclosed protease activity sensor and biocomparator can be used to distinguish between programmed cell death, which indicates an immune response, and necrosis, which occurs naturally during tumor progression. In contrast to programmed cell death, in which caspases and serine proteases are the main proteases, calpains and lysosomal proteases (e.g., cathepsins B and D) are key proteases in necrosis. Therefore, the calpain and cathepsin levels indicated by activity sensor reporter measurements can provide information about necrotic cell death to complement cancer immunological information.

[0048] The protease activity sensor can release a reporter molecule upon cleavage by two different proteases. The detectable signal can be a cleavage product or a peptide fragment of the protease substrate itself. Upon cleavage, fragments of the protease substrate are released into the circulation and detected in urine by mass spectrometry. Cleavage by one protease can release a reporter molecule. However, a detectable signal cannot be detected from the released reporter molecule until cleavage by a second protease. Such a second cleavage can, for example, cleave a fluorescent quencher from the reporter molecule, thereby enabling detection. Alternatively, the detectable signal is a protease substrate engineered with a quencher molecule before the cleavage site(s) and a fluorescent reporter molecule after the cleavage site(s). Upon cleavage of the protease substrate, the quencher and fluorescent reporter are separated, and the reporter is released into the circulation. The fluorescent signal is detected in urine by standard methods, such as flow cytometry.

[0049] The protease substrate can be conjugated to a reporter molecule, a quencher, and / or a scaffold using methods known in the art. In one embodiment, the protease substrate is conjugated by introducing a linker that forms a covalent conjugate between the protease substrate and the reporter molecule, the quencher, and / or the scaffold. Representative reactions that can be used to link the protease substrate include, but are not limited to, amine-amine crosslinkers using NHS esters, thiol-thiol crosslinkers using maleimides, amine-thiol crosslinkers using NHS esters and maleimides, and biotin / streptavidin interactions.

[0050] The reporter molecule released from the active sensor of the present invention can be detected by any suitable detection method that can directly or indirectly detect the presence of the reporter molecule.For example, the reporter can be detected through a ligand binding assay, which is a test that involves the binding of a capture ligand to an affinity agent.After capture, the reporter can be directly detected through optical density, radioactive emission, or non-radioactive energy transfer.Alternatively, the reporter can be indirectly detected using an antibody conjugate, an affinity column, a streptavidin-biotin conjugate, PCR analysis, DNA microarray, or fluorescence analysis.

[0051] Ligand binding assays often include a detection step such as an ELISA, including fluorescent, colorimetric, bioluminescent and chemiluminescent ELISAs, paper test strips or lateral flow assays, or bead-based fluorescent assays.

[0052] In one example, a paper-based ELISA test can be used to detect free reporters in urine. Paper-based ELISAs can be produced inexpensively, such as by reflowing wax deposited from a commercially available 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, creating a hydrophobic barrier. The spaces between the hydrophobic barriers can then be used as individual reaction wells. The ELISA assay can be performed by drying a detection antibody onto each reaction well, forming test spots on the paper, followed by a blocking and washing step. Urine from a urine sample collected from a subject is then added to the test spots, followed by the addition of a streptavidin alkaline phosphate (ALP) conjugate as the detection antibody. The bound ALP can then be exposed to a color-developing reagent, such as BCIP / NBT (5-bromo-4-chloro-3'-indolylphosphate p-toluidine salt / nitro-blue tetrazolium chloride), which causes a purple precipitate, indicating the presence of the reporter molecule.

[0053] In other examples, volatile organic compounds can be detected by analytical platforms such as gas chromatography instruments, breath analyzers, mass spectrometers, or the use of optical or acoustic sensors.

[0054] Gas chromatography can be used to detect compounds that can be vaporized 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 gaseous compounds being analyzed interact with the column walls, causing them to elute at different times (i.e., have different retention times in the column). Compounds can be distinguished by their retention times.

[0055] Improved breath analysis devices can also be used to detect volatile organic compounds. In traditional breath analyzers used to detect blood alcohol levels, a subject exhales into the device, and ethanol present in the subject's breath is oxidized to acetic acid at the anode. Atmospheric oxygen is reduced at the cathode. The overall reaction is the oxidation of ethanol to acetic acid and water, which results in a current that can be detected and quantified by a microcontroller. Improved breath analysis devices that utilize other reactions may also be used to detect various volatile organic compounds.

[0056] Mass spectrometry can be used to detect and identify reporters based on mass differences. In mass spectrometry, a sample is ionized, for example, by bombarding it with electrons. The sample can be solid, liquid, or gas. By ionizing a 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 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. Once deflected, the ions can be detected by a mechanism that can detect charged particles, such as an electron multiplier. The detection result can be shown as a spectrum of the relative abundance of detected ions as a function of mass-to-charge ratio. Then, molecules in a sample can be identified by correlating known masses, such as the mass of the entire molecule, with the identified masses, or through characteristic fragmentation patterns.

[0057] When the reporter comprises nucleic acid, the reporter can be detected by various sequencing methods known in the art, for example, by traditional Sanger sequencing method or by next-generation sequencing (NGS).NGS generally refers to non-Sanger-based high-throughput nucleic acid sequencing technology, which can determine the sequence of many (i.e., thousands, millions, or billions) nucleic acid strands in parallel.Examples of such NGS sequencing include the platforms produced by Illumina (e.g., HiSeq, MiSeq, NextSeq, MiniSeq, and iSeq 100), Pacific Biosciences (e.g., Sequel and RSII), and ThermoFisher's Ion Torrent (e.g., Ion S5, Ion Proton, Ion PGM, and Ion Chef system).It is understood that any suitable NGS sequencing platform can be used for NGS to detect the nucleic acid of reporter molecule as described herein.

[0058] Analysis can be performed directly on the biological sample, or the reporter molecule may first be purified to some extent. For example, the purification step can include isolating the reporter molecule from other components in the biological sample. Purification can include methods such as affinity chromatography. The isolated or purified reporter molecule need not be 100% pure or substantially pure prior to analysis.

[0059] The reporter molecule may be attached to a label or may itself contain a label. Labels suitable for use in reporter molecules include any type of label that can be detected by standard methods, such as spectroscopic, photochemical, biochemical, electrical, optical, or chemical methods. The label can be a fluorescent label. A fluorescent label is a compound that contains at least one fluorophore. Commercially available fluorescent labels include, for example, fluorescein phosphoramidite, rhodamine, polymethadine dye derivatives, phosphores, Texas Red, green fluorescent protein, CY3, and CY5. Other known techniques, such as chemiluminescence or colorimetric methods (enzymatic color reactions), can also be used to detect reporters. Quencher compositions in which a "donor" fluorophore is joined to an "acceptor" chromophore via a short bridge that serves as the binding site for the enzyme, can also be used. The signal of the donor fluorophore is quenched by the acceptor chromophore through a process believed to involve resonance energy transfer (RET), such as fluorescence resonance energy transfer (FRET). Cleavage of the peptide results in separation of the chromophore and fluorophore, removal of quenching, and generation of the subsequent signal measured from the donor fluorophore. Examples of FRET pairs include 5-carboxyfluorescein (5-FAM) and CPQ2, FAM and DABCYL, Cy5 and QSY21, and Cy3 and QSY7.

[0060] The reporter molecule can comprise one or more protease substrates engineered with a quencher molecule before the cleavage site(s) and a fluorophore or fluorescent reporter after the cleavage site(s). Quencher molecules are known in the art. Exemplary quencher molecules include, but are not limited to, Deep Dark Quenchers (Eurogentec), DABCYL, TAMRA, BHQ-1®, BHQ-2®, BHQ-3®, BBQ®-650, ECLIPSE, Iowa Black® quencher, and QSY. Exemplary fluorophores or fluorescent reporters include, but are not limited to, 6-FAM. TM , TET TM , JOE TM , HEX TM , VIC®, Cyanine 3, ROX TM , LC Red 640, Cyanine 5, fluorescein isothiocyanate (FITC), rhodamine (tetramethylrhodamine isothiocyanate, TRITC), Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, Texas Red, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, and Alexa Fluor 750.

[0061] The protease activity sensor or biocomparator may contain other reporter molecules, such as avidin, biotin, beta-galactosidase, luciferase, alkaline phosphatase (AP), and horseradish peroxidase (HRP). The reporter molecule can be cleaved from the protease substrate and released into the circulation. The reporter molecule can be released from the biocomparator and released into the circulation. The reporter molecule is then detected in the urine sample using a suitable detection method, such as, but not limited to, ELISA, Western blotting, immunoassay, and bioluminescence assay.

[0062] Protease activity sensors or biocomparators can include ligands to assist them in targeting specific tissues or organs. When administered to a subject, protease activity sensors or biocomparators can be transported through the body via various routes depending on how they enter the body. For example, when administered intravenously, they can enter the systemic circulation from the point of injection and be passively transported throughout the body.

[0063] The protease activity sensor or bicomparator can include a scaffold. The scaffold can further include a tuning domain or can be conjugated to the tuning domain. The tuning domain can modify the distribution or residence time of the protease activity sensor or bicomparator within a subject when administered to the subject. The protease activity sensor or bicomparator can be tuned in numerous ways via the tuning domain to facilitate detection of enzyme activity in specific cells or tissues within the body. For example, the protease activity sensor or bicomparator can be tuned to promote distribution to specific tissues or to improve residence time in a subject or specific tissue. The tuning domain can include a molecule localized in rapidly replicating cells, for example, to better target tumor tissue.

[0064] Upon administration to a subject, the protease activity sensor or biocomparator is transported throughout the body and can diffuse from the systemic circulation to specific tissues where the reporter can be released via the enzyme, indicating cancer progression or an immune response. The reporter molecule then diffuses back into the circulation, where it can pass through renal filtration and be excreted in the urine, such that detection of the reporter molecule in a urine sample indicates enzyme activity in the target tissue.

[0065] The scaffold can be any suitable platform for delivering the protease activity sensor or biocomparator into the subject's body. The scaffold can be of any material or size suitable for serving as a scaffold or platform. Preferably, the scaffold is biocompatible, non-toxic, and non-immunogenic, and does not induce an immune response in the subject to which it is administered. The scaffold can also serve as a targeting tool for targeting the protease activity sensor or biocomparator to tissues, cells, or molecules. The scaffold can be a polymer scaffold. The scaffold can provide passive targeting, for example, to tumors or other specific tissues via the circulation. Other types of scaffolds include, for example, compounds that promote active targeting to tissues, cells, or molecules. Examples of scaffolds include, but are not limited to, nanoparticles such as iron oxide or gold nanoparticles, aptamers, peptides, proteins, nucleic acids, polysaccharides, polymers, antibodies or antibody fragments, and small molecules.

[0066] Scaffolds can include a variety of materials, such as iron, ceramics, metals, natural polymeric materials such as hyaluronic acid, synthetic polymeric materials such as polyglycerol sebacate, and non-polymeric materials, or combinations thereof. Scaffolds can be composed entirely or partially of polymeric or non-polymeric materials, such as alumina, calcium carbonate, calcium sulfate, calcium phosphosilicate, sodium phosphate, calcium aluminate, and silicates. Polymers include, but are not limited to, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, cellulose ethers, cellulose esters, nitrocellulose, polymers of acrylic and methacrylic acid esters, methylcellulose, ethylcellulose, and hydroxypropylcellulose. Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers, and mixtures thereof.

[0067] Examples of biodegradable polymers include synthetic polymers, such as polymers of lactic acid and glycolic acid, polyanhydrides, polyurethanes, and natural polymers, such as alginates and other polysaccharides, such as dextran and cellulose, collagen, albumin and other proteins, copolymers, and mixtures thereof. Generally, these biodegradable polymers degrade by either enzymatic hydrolysis or exposure to water in vivo, through surface or bulk erosion. These biodegradable polymers can be used alone, as physical mixtures (blends), or as copolymers. In a preferred embodiment, the scaffold comprises a biodegradable polymer such that the scaffold degrades in the body regardless of whether the reporter is cleaved from the scaffold. By providing a biodegradable scaffold, the accumulation of remaining intact active sensors in the body and any associated immune responses or unintended effects can be minimized.

[0068] Other biocompatible polymers include PEG, PVA, and PVP, all of which are commercially available. PVP is a non-ionogenic hydrophilic polymer with an average molecular weight ranging from approximately 10,000 to 700,000 and has the chemical formula (C6H9NO)[n]. PVP is also known as poly[1(2-oxo-1-pyrrolidinyl)ethylene]. PVP is non-toxic, highly hygroscopic, and readily soluble in water or organic solvents.

[0069] Polyvinyl alcohol (PVA) is a polymer prepared from polyvinyl acetate by replacing the acetate groups with hydroxyl groups and has the chemical formula (CHCHOH)[n]. Most polyvinyl alcohols are soluble in water.

[0070] Polyethylene glycol (PEG), also known as poly(oxyethylene) glycol, is a condensation polymer of ethylene oxide and water. PEG refers to compounds containing ethylene glycol repeating units. The structure of PEG can be represented as H—(O—CH—CH)—OH. PEG is a hydrophilic compound that is biologically inert (i.e., non-immunogenic) and generally considered safe for human administration.

[0071] When PEG is linked to particles, it offers advantageous properties such as improved solubility, increased circulation life, stability, protection from proteolysis, reduced cellular uptake by macrophages, and a lack of immunogenicity and antigenicity. PEG is also highly flexible, allowing for particle bioconjugation and surface treatment without steric hindrance. PEG can be used to chemically modify biologically active compounds, such as peptides, proteins, antibody fragments, aptamers, enzymes, and small molecules, to tailor the molecular properties of the compound to specific applications. Furthermore, PEG molecules can be functionalized by chemically attaching various functional groups to the termini of the PEG molecule, such as amine-reactive PEG (BS(PEG)n) or sulfhydryl-reactive PEG (BM(PEG)n).

[0072] The scaffold can be a biocompatible scaffold, such as one containing polyethylene glycol (PEG). The biocompatible scaffold can contain multiple covalently attached polyethylene glycol maleimide (PEG-MAL) subunits, e.g., an 8-arm PEG-MAL scaffold. PEG-containing scaffolds can be selected because they are biocompatible, inexpensive, readily available commercially, exhibit minimal uptake by the reticuloendothelial system (RES), and exhibit many advantageous behaviors. For example, PEG scaffolds inhibit cellular uptake of particles by many cell types, such as macrophages, thereby facilitating proper distribution to specific tissues and increasing retention time in tissues.

[0073] The 8-arm PEG-MAL is a multi-arm PEG derivative with eight arms attached to a hexaglycerol core, each with a maleimide group at its terminus. The maleimide groups selectively react with free thiol, SH, sulfhydryl, or mercapto groups via Michael addition to form stable carbon-sulfur bonds. Each arm of the 8-arm PEG-MAL scaffold can be conjugated to a peptide via, for example, a maleimide-thiol or amide bond.

[0074] The PEG-MAL scaffold can be of various sizes, for example, a 10 kDa scaffold, a 20 kDa scaffold, a 40 kDa scaffold, or a scaffold larger than 40 kDa. The hydrodynamic diameter of the PEG scaffold in phosphate buffered saline (PBS) can be determined by various methods known in the art, for example, dynamic light scattering. Using this technique, the hydrodynamic diameter of a 40 kDa PEG-MAL scaffold was measured to be about 8 nm. In a preferred embodiment, a 40 kDa PEG-MAL scaffold is used as a scaffold when the protease activity sensor or biocomparator is administered subcutaneously. This is because the scaffold easily diffuses into the systemic circulation but is not easily removed by the reticuloendothelial system.

[0075] The size of the PEG-MAL scaffold influences the distribution and residence time of the protease activity sensor or biocomparator in the body. This is because particles smaller than approximately 5 nm in diameter are efficiently removed by the body's renal filtration, even in the absence of proteolytic cleavage. Furthermore, particles larger than approximately 10 nm in diameter are often excreted into the lymphatic system. In one example, when a 40 kDa 8-arm PEG-MAL scaffold was administered intravenously, the scaffold was not excreted in the urine by the kidney.

[0076] Protease activity sensors or biocomparators can include cyclic peptides that are structurally resistant to nonspecific proteolysis and degradation in the body. Cyclic peptides can include protease-specific substrates or pH-sensitive bonds that allow otherwise unreactive cyclic peptides to release reactive reporter molecules in response to the presence of enzymes discussed herein. Cyclic peptides may require cleavage at multiple cleavage sites to increase specificity. The multiple sites can be specific for different proteases. Polycyclic peptides containing two, three, four, or more cyclic peptide structures can be used with various combinations of enzymes or environmental conditions required to linearize or release functional peptides or other molecules. Cyclic peptides can include depsipeptides, where hydrolysis of one or more ester bonds releases the linearized peptide. Such peptides can be used to time peptide release in environments such as plasma.

[0077] Figure 8 shows a representative protease activity sensor comprising a protease-specific substrate 809 and a cyclic peptide 801 with a stable cyclization linker 803. Each protease-specific substrate 809 is cleaved by a different protease and can contain any number of amino acids in any order. For example, X1 can be glycine, X2 can be serine, X3 can be aspartic acid, X4 can be phenylalanine, X5 can be glutamic acid, and X6 can be isoleucine. These amino acids can vary between individual substrates to provide specificity for different proteases. The N- and C-termini attached to the cyclization linker 803 contain cyclization residues 805. The peptide can be engineered to address considerations such as protease stability, steric hindrance around the cleavage site, macrocyclic structure, and rigidity / flexibility of the peptide chain. The type and number of spacer residues 807 can be chosen so that many of these properties can be addressed and modified by varying the spacing between the various functional sites of the cyclic peptide. The positioning and selection of the cyclization linker, as well as the cyclization residue, may also affect the above considerations. Tuning domains such as PEG, reporters such as FAM, and quenchers can be incorporated into cyclic peptides.

[0078] The tuning domain can include a ligand to assist in targeting to a specific tissue or organ. When administered to a subject, protease activity sensors or biocomparators are transported through the body via various pathways depending on how they enter the body.

[0079] Cell surface receptors are membrane-anchored proteins that bind to ligands on the outer surface of a cell. In one example, a ligand can bind to a ligand-gated ion channel, an ion channel that opens in response to ligand binding. Ligand-gated ion channels span the membrane of a cell and have a hydrophilic channel in the middle. In response to ligand binding to the extracellular region of the channel, the protein's structure changes to allow specific particles or ions to pass through. By providing a tuning domain that encompasses the ligand of a protein present on the cell surface, a protease activity sensor or biocomparator has a greater opportunity to reach and enter specific cells and detect enzyme activity within those cells.

[0080] By providing a tuning domain, the distribution of the protease activity sensor or biocomparator can be modified. This is because the ligand can target specific cells or specific tissues of a subject through binding of the ligand to cell surface proteins on the target cells. The ligand of the tuning domain can be selected from the group including small molecules; peptides; antibodies; antibody fragments; nucleic acids; and aptamers. The ligand can also promote the accumulation of the protease activity sensor or biocomparator in specific tissue types.

[0081] When protease activity sensors or biocomparators are administered to a subject, they may be recognized as foreign by the immune system and subject to immune clearance, preventing them from reaching specific cells or tissues where specific enzyme activity can release reporter molecules. Furthermore, the generation of an immune response may defeat the purpose of immune response-sensitive activity monitoring. To inhibit immune detection, it is preferable to use a biocompatible scaffold that does not induce an immune response. For example, the biocompatible scaffold may include one or more polyethylene glycol maleimide subunits. Furthermore, modifying the molecular weight of the polyethylene glycol maleimide scaffold can facilitate transport within the body and prevent clearance by the reticuloendothelial system. Such modifications can improve distribution and residence time in the body or in specific tissues.

[0082] In various embodiments, the protease activity sensor or biocomparator can be engineered to facilitate diffusion across the cell membrane. Hydrophobic chains can also serve as tuning domains to facilitate diffusion across the cell membrane.

[0083] The tuning domain can include any suitable hydrophobic chain that promotes diffusion, such as fatty acid chains, including neutral, saturated, (poly / mono)unsaturated fats and oils (monoglycerides, diglycerides, triglycerides), phospholipids, sterols (steroid alcohols), zoosterols (cholesterol), waxes, and fat-soluble vitamins (vitamins A, D, E, and K).

[0084] The tuning domain can include a cell-penetrating peptide. Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular uptake / intake. CPPs preferably have an amino acid composition that contains a high relative abundance of positively charged amino acids, such as lysine or arginine, or a sequence containing an alternating pattern of polar / charged amino acids and non-polar hydrophobic amino acids. See Milletti, 2012, Cell-penetrating peptides: classes, origin, and current landscape, Drug Discovery Today 17:850-860, which is incorporated by reference. Suitable CPPs include those known in the literature as Tat, R6, R8, R9, Penetratin, pVEc, RRL helix, Shuffle, and Penetramax. See Kristensen, 2016, Cell-penetrating peptides as tools to enhance non-injectable delivery of biopharmaceuticals, Tissue Barriers 4(2):e1178369, incorporated by reference.

[0085] The protease activity sensor or biocomparator can include a biocompatible polymer as a tuning domain to shield the activity sensor from immunodetection or inhibit cellular uptake of the activity sensor by macrophages.

[0086] When a foreign substance is recognized as an antigen, an antibody response can be elicited by the immune system. Generally, antibodies then attach to the foreign substance to form antigen-antibody complexes, which are then ingested by macrophages and other phagocytes to remove these foreign substances from the body. Therefore, when protease activity sensors or biocomparators enter the body, they can be recognized as antigens and subjected to immune clearance, preventing them from reaching specific tissues and detecting biomarker activity. To inhibit immune detection, for example, a PEG tuning domain can be linked to the activity sensor. PEG acts as a shield, inhibiting recognition as a foreign substance by the immune system. By inhibiting immune detection, the tuning domain improves residence time in the body or in specific tissues.

[0087] Enzymes have high specificity for specific substrates due to the binding pockets that have complementary shape, charge, and hydrophilic / hydrophobic properties of the substrate. Thus, enzymes can distinguish between very similar substrate molecules such that they are chemoselective (i.e., preferring the outcome of a chemical reaction over alternative reactions), regioselective (i.e., preferring one direction of chemical bond formation or breaking over all other possible directions), and stereospecific (i.e., reacting only to one or a subset of stereoisomers).

[0088] Steric effects are non-bonded interactions that affect the shape (i.e., conformation) and reactivity of ions and molecules, resulting in steric hindrance. Steric hindrance is a slowing down of chemical reactions due to steric bulk, affecting intermolecular reactions. Various groups on molecules can be modified to control steric hindrance between groups, thereby controlling selectivity, for example, to inhibit undesired side reactions. Providing a protease activity sensor or biocomparator with tuning domains, such as spacer residues between the scaffold and the cleavage site and / or any bioconjugation residues, can minimize steric hindrance between components and increase the accessibility of the cleavage site(s) for a particular protease.

[0089] Alternatively, steric hindrance can be used as described above to prevent access to the cleavage site(s) until a labile cyclization linker (e.g., an ester bond in a cyclic depsipeptide) is degraded. Such labile cyclization linkers can be other known chemical moieties that hydrolyze under defined conditions (e.g., pH or the presence of a particular analyte) that can be selected to respond to specific properties of the target environment.

[0090] The protease activity sensor or biocomparator can include D-amino acids in addition to the protease cleavage site(s) to further prevent non-specific protease activity. Other non-natural amino acids, such as synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids, can also be incorporated into the peptide.

[0091] In some embodiments, tuning domains can include synthetic polymers, such as polymers of lactic and glycolic acid, polyanhydrides, polyurethanes, and natural polymers, such as alginate and other polysaccharides, e.g., dextran and cellulose, collagen, albumin and other hydrophilic proteins, zein and other prolamins and hydrophobic proteins, copolymers, and mixtures thereof.

[0092] Those skilled in the art will know which peptide segments should be included as protease substrates / cleavage sites in the protease activity sensors or biocomparators of the present disclosure. Online tools or publications can be used to identify protease substrates / cleavage sites. For example, cleavage sites are predicted in the online tool PROSPER, as described in Song, 2012, PROSPER: An integrated feature-based tool for predicting protease substrate cleavage sites, PLoSOne 7(11):e50300, which is incorporated by reference. Any of the compositions, structures, methods, or activity sensors discussed herein can include, for example, any suitable cleavage site, as well as any additional polypeptide segments, to achieve any desired molecular weight. To prevent off-target cleavage, one or any number of amino acids outside the cleavage site can be in a mixture of D and / or L forms in any amount.

[0093] The biological sample can be any sample from a subject in which the reporter can be detected. For example, the sample can be a tissue sample (e.g., a blood sample, a hard tissue sample, a soft tissue sample, etc.), a urine sample, a saliva sample, a mucus sample, a feces sample, a semen sample, or a cerebrospinal fluid sample.

[0094] Pharmaceutical compositions containing the disclosed protease activity sensors or bicomparators are disclosed. Pharmaceutical compositions containing the protease activity sensors or bicomparators can be administered parenterally (intramuscularly, intraperitoneally, intravenously (IV), or subcutaneously), transdermally (passively, or using iontophoresis or electroporation), or transmucosally (nasally, vaginally, rectally, or sublingually), or using bioerodible inserts, and can be formulated in dosage forms suitable for each administration route. The exact dosage will vary according to various factors, including subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being performed.

[0095] Compositions disclosed herein, including those containing peptides and polypeptides, are administered parenterally in aqueous solution. Formulations can also be in the form of suspensions or emulsions. Generally, pharmaceutical compositions are provided that contain an effective amount of a peptide or polypeptide and may include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions may include one or more of the following: diluents, sterile water, buffered saline solutions of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; and additives, such as detergents and solubilizers (e.g., TWEEN® 20 (polysorbate-20), TWEEN® 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid), and preservatives (e.g., Thimersol, benzyl alcohol), and bulking agents (e.g., lactose, mannitol). Examples of non-aqueous solvent or vehicle are propylene glycol, polyethylene glycol, vegetable oil (e.g., olive oil and corn oil), gelatin, and injectable organic esters such as ethyl oleate.Preparation can be lyophilized and redissolved / resuspended immediately before use.Preparation can be sterilized, for example, by filtering through a bacteria-retaining filter, by incorporating sterilizing agent into composition, by irradiating composition, or by heating composition.

[0096] In some embodiments, the composition is formulated for oral delivery. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules, or the incorporation of materials into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate. The composition may be prepared in liquid form or in dry powder (e.g., lyophilized) form. The composition may be formulated using liposome or proteinoid encapsulation. Liposomal encapsulation may be used, and liposomes may be derivatized with various polymers (e.g., Patent No. 5013556). Generally, the formulation includes a peptide (or a chemically modified form thereof) and an inactive component that protects the peptide in the stomach environment and releases the biologically active substance in the intestine.

[0097] Drugs can be chemically modified to enable effective oral delivery of derivatives. Generally, the intended chemical modification is the attachment of at least one moiety to the component molecule itself, where the moiety allows uptake into the bloodstream from the stomach or intestine, or allows direct uptake into the intestinal mucosa. Improved overall stability of the component or components and increased circulation time in the body are also desirable. PEGylation is a typical chemical modification for pharmaceutical use. Other moieties that can be used include propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane, and poly-1,3,6-tioxocane.

[0098] Other embodiments provide liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other ingredients, such as inert diluents; adjuvants such as wetting agents, emulsifying agents and suspending agents; and sweetening, flavoring, and perfuming agents.

[0099] Controlled release oral formulations may be desirable. The drug can be incorporated into an inert matrix, such as gum, which allows release by either diffusion or leaching mechanisms. Slowly degenerating matrices can also be incorporated into the formulation. Another form of controlled release is based on the Oros Therapeutic System (Alza Corp.), i.e., the drug is encapsulated in a semipermeable membrane, which allows water to enter through a single small opening and push the drug out by osmotic effect.

[0100] For oral formulations, the location of release can be the stomach, the small intestine (duodenum, jejunum, or ileum), or the large intestine. In some embodiments, release avoids the harmful effects of the stomach environment, either by protecting the drug (or derivative) or by releasing the drug (or derivative) beyond the stomach environment, e.g., in the intestine. To ensure complete gastric resistance, a coating impermeable to at least pH 5.0 is essential. Examples of more common inactive components used as enteric coatings include cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D. TM , Aquateric TM , Cellulose acetate phthalate (CAP), Eudragit L TM , Eudragit S TM , and Shellac TM These coatings may also be used as mixed films.

[0101] The disclosed immunotherapeutics can be applied locally, which does not work well for most peptide formulations, but can be effective, particularly when applied to the mucous membranes of the lungs, nose, mouth (sublingual, buccal), vagina, or rectum.

[0102] When the composition is delivered as either an aerosol or spray-dried particles with an aerodynamic diameter of less than about 5 microns, it can be delivered to the lungs during inhalation and cross the pulmonary epithelial layer to the bloodstream.A wide range of mechanical devices designed for pulmonary delivery of therapeutic agents can be used, including but not limited to nebulizers, metered-dose inhalers, and powder inhalers, all of which are well known to those skilled in the art.Formulations for administration to mucosa are typically spray-dried drug particles, which can be incorporated into tablets, gels, capsules, suspensions, or emulsions.Standard pharmaceutical excipients can be obtained from any formulation manufacturer.

[0103] Transdermal formulations can also be prepared. These are typically ointments, lotions, sprays, or patches, all of which can be prepared using standard techniques. Transdermal formulations may require the inclusion of penetration enhancers.

[0104] The biological sample can be any sample from a subject in which a reporter can be detected. For example, the sample can be a tissue sample (e.g., a blood sample, a hard tissue sample, a soft tissue sample, etc.), a urine sample, a saliva sample, a mucus sample, a feces sample, a semen sample, or a cerebrospinal fluid sample. In some aspects, the sample can be obtained from the subject's tissue or bodily fluid, or from a swab taken from the patient. Samples can include fine needle aspirates, biopsies, or bodily fluids from the patient. The sample can be processed, for example, to produce a suspension containing an appropriate solution. Such solutions are generally balanced salt solutions, such as saline, PBS, Hank's balanced salt solution, and the like, and in certain cases are supplemented with fetal bovine serum or other naturally occurring factors at low concentrations, generally 5-25 mM, along with an acceptable buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, and the like. In a preferred embodiment, the sample is a respiratory swab (e.g., an oral swab, a nasal swab, or a throat swab). The swab can be placed in a sterile tube containing a medium (e.g., Hank's balanced salt solution). The medium may also contain an antibiotic to reduce the possibility of bacterial contamination. [Example]

[0105] Example 1: Granzyme B / thrombin AND gate protease activity sensor Figures 9-14 show AND-gated sensors that sense the proteases granzyme B (GzmB) and thrombin (Thrb), which play roles in target cell killing by the immune system and blood coagulation, respectively. These AND-gated protease sensors are designed to release a fluorescent reporter (5-FAM) only after cleavage of both protease substrates (Figure 9). Figure 9 shows that the protease activity sensor contains a dark quencher (DABCYL), a fluorescent reporter (5-FAM), substrates for both GzmB and Thrb (bold, cleavage sites indicated by "|" symbols), and an azide for click conjugation to scaffolds such as nanoparticles. In the absence of both proteases, a disulfide bridge cyclizes the peptide, ensuring quenching of 5-FAM. When the AND condition is met, i.e., the state represented by "1,1," 5-FAM fluoresces.

[0106] To validate the sensor design, the specificity of the protease substrates was tested to ensure that each substrate could be cleaved by only one of the selected proteases. Individual protease substrates were labeled with a fluorescent quencher and dye. Cleavage assays were performed by incubating the substrates with GzmB or Thrb and monitoring fluorescence. Indeed, the GzmB substrate was efficiently cleaved by GzmB alone (Figure 10), and the Thrb substrate was cleaved only by Thrb (Figure 11). Figures 10-11 show the average change in fluorescence after 1.5 h incubation of nanoparticle-conjugated GzmB substrates (450 nM) and Thrb substrates (330 nM) with no protease, 275 nM GzmB, or 33 nM Thrb, respectively (n = 3).

[0107] Next, the AND-gate logic protease sensor was tested in a cleavage assay. The reporter molecule emitted a high fluorescence signal in the presence of both proteases together, but not in the presence of either protease alone (Figure 12). This indicates that a detectable signal was present only upon cleavage by both proteases. Figure 12 shows kinetic traces of the average change in fluorescence during 12-hour incubation of a nanoparticle-conjugated GzmB / Thrb AND-gate sensor (1 μM) with 125 nM GzmB and / or 3.7 nM Thrb (n = 2).

[0108] Figures 13-14 show that the AND gate logic sensor is highly sensitive to small changes in the concentration of both proteases, even in the nanomolar range. Therefore, it can detect even small amounts of protease when other proteases are also present. Figures 13-14 show the average change in fluorescence after 2 hours of incubation of the AND gate sensor with 9.2 nM Thrb and various concentrations of GzmB (0-150 nM) (Figure 13) or 300 nM GzmB and various concentrations of Thrb (0-4.6 nM) (Figure 14) (n=2). For all fluorescence readouts, samples were excited at 485 nm and emission was measured at 528 nm. Samples were incubated at 37°C.

[0109] Example 2: AND-gated protease activity sensor in biological systems Figures 15-17 show examples of the disclosed AND-gate protease activity sensor used with biological systems. The sensor was evaluated in an in vitro transgenic T cell killing assay in which cytotoxic T cells collected from OT1 mice were co-incubated with target cells EL4 and EG7-OVA (Figure 15). Because OT1 T cells can recognize the OVA antigen and initiate an immune response, the T cells theoretically would respond to EG7-OVA cells but not EL4 cells. Supernatants were collected from the co-incubations, and the immune response via GzmB secretion was quantified using ELISA (Figure 16). After overnight co-incubation with target cells at 37°C, concentrations were measured by ELISA (n=3). It was confirmed that co-incubation of OT1 cells with EG7-OVA cells increased GzmB secretion by OT1 cells but not by EL4 cells.

[0110] Next, the supernatant was used as a GzmB source, and Thrb was added to perform a cleavage assay using an AND-gate sensor. Figure 17 shows kinetic traces of the mean change in fluorescence during 12 h incubation, co-incubation, and / or 3.7% Thrb of the nanoparticle-conjugated GzmB / Thrb AND-gate protease activity sensor (1 μM) with 10% supernatant from EL4 and EG7-OVA (n=3). Data for EL4 and EG7-OVA were normalized by subtracting traces using OT1-only supernatant, and data for EL4 + Thrb and EG7-OVA + Thrb were normalized by subtracting traces using OT1-only supernatant and Thrb. A high signal was achieved only when Thrb was added to the GzmB-high OT1 / EG7-OVA supernatant, demonstrating that the AND-gate protease activity sensor can accurately probe biological activity based on Boolean logic.

[0111] Example 3: AND-gated protease activity sensor for monitoring efficacy and response to therapeutic treatment Figures 18-21 show an AND-gated protease sensor for monitoring efficacy and response to therapeutic treatment. Because cancer immunotherapy is limited by its off-target effects, noninvasive detection of intratumoral immune activity substantially improves treatment monitoring. In addition, simply monitoring a patient's immune activity does not confirm the efficacy of immunotherapy because the immune system may be activated against other pathologies (e.g., viral infections). Therefore, the AND-gated logic sensor can improve the specificity of treatment monitoring by sensing both GzmB and tumor-associated proteases. Matrix metalloproteinases (MMPs) are a type of protease that are highly upregulated in many types of cancer due to their role in extracellular matrix remodeling, which aids tumor growth, invasion, and metastasis.

[0112] Figure 18 shows an AND-gate protease activity sensor that probes both GzmB and MMPs. MMP substrates were designed based on the literature and tested to ensure that the substrates could be cleaved by various pure MMPs (Figure 19). Figure 19 shows kinetic traces of the average change in fluorescence during a 75-minute incubation of the MMP substrate (5 μM) with 100 nM of various MMPs (n=2). The substrate was cleaved with high activity by MMP1, MMP8, and MMP13.

[0113] We then performed cleavage assays using the GzmB / MMP AND gated sensor, testing the probe with each MMP, with and without GzmB (Figure 20). For all three MMPs, the addition of GzmB dramatically increased the fluorescent signal by more than two-fold, confirming that the probe follows the desired Boolean logic.

[0114] Next, we incubated the protease activity sensor with GzmB and MMP8 or one of two viral proteases (West Nile virus NS3 protease and tobacco etch virus protease) to demonstrate that the AND-gated sensor can distinguish immune activity in tumors from viral infection (Figure 21). The sensor produced a lower signal with the viral proteases compared to MMP8.

[0115] Figures 20-21 show the average change in fluorescence (7D, kinetic traces) after 1.5 h incubation of the GzmB / MMP AND gate sensor (5 μM) with (7C) 300 nM GzmB and / or 100 nM of various MMPs, or (7D) 300 nM GzmB and 100 nM of West Nile virus NS3 protease, tobacco etch virus protease, or MMP8 (n=3).

[0116] Example 4: Representative AND-Gated Biocomparator The concept of an AND gate logic sensor system is not limited to implementation with a protease activity sensor. This disclosure provides an AND gate biocomparator consisting of a liposome in a peptide cage formed by an MMP substrate. Because the liposome encapsulated a fluorescent reporter, the peptide cage had to be cleaved, and the liposome had to be perforated by perforin, a cytolytic protein used by T cells, to release the reporter. The results show that by placing a fluorescent reporter in the liposome, perforin can perforate the liposome, and the fluorescent reporter is released upon perforation (Figure 22). The results also confirmed that MMP9 can cleave the MMP substrate by a cleavage assay (Figure 22). Figure 22 shows a fluorescent assay for detecting the opening of naked liposomes by perforin (top panel) and the cleavage of the peptide substrate by the protease MMP9 (bottom panel).

[0117] Next, the peptide-caged biocomparator was tested with perforin, MMP9, or both proteins (Figure 23). Only both proteins resulted in a high signal, suggesting that both are required to release the reporter in the biocomparator. Figure 23 shows a fluorescence assay measuring the increase in signal from a biocomparator opened by both a signal protease (MMP9) and perforin.

[0118] Finally, results from a T cell killing assay demonstrated that the biocomparator could be opened only when there was an immune response and MMP9 was present. Figure 24 shows an experimental schematic depicting the production of MMP9 and perforin in the context of antigen-specific killing. T cells were collected from OT1 mice and co-incubated with target tumor cells (EG7) and off-target cells that secrete MMP9. Figure 25 shows a T cell killing assay measuring an increase in signal from the biocircuit, which can detect both T cell killing and tumor activity.

[0119] While the invention has been described in the foregoing specification with reference to specific embodiments thereof, and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is capable of additional embodiments and that considerable variation can be made in the specific details described herein without departing from the underlying principles of the invention.

[0120] All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from its spirit or essential attributes, and therefore, reference should be made to the appended claims, rather than the foregoing specification, as indicating the scope of the invention. This specification includes the disclosure of the following inventions. [Item 1] A method for determining protease activity in a biological sample, comprising: providing a protease activity sensor in a biological sample, the protease activity sensor comprising a first protease substrate and a second protease substrate, wherein the first substrate is cleaved by the first protease and the second substrate is cleaved by the second protease, and wherein the protease sensor provides a detectable signal when both the first substrate and the second substrate are cleaved; detecting the presence of a detectable signal in the sample; and identifying the activity of the first protease and the second protease based on the presence of a detectable signal in the sample; The method comprising: [Item 2] The method according to Item 1, wherein the protease activity sensor is conjugated to a reporter molecule that provides a detectable signal. [Item 3] The method according to Item 2, wherein the reporter molecule is a fluorescent molecule. [Item 4] The method of item 3, wherein the first substrate and the second substrate are conjugated to a fluorescent quencher, and cleavage of the first substrate and the second substrate enables the fluorescent molecule to provide a detectable signal. [Item 5] The method according to Item 1, wherein the protease activity sensor is a cyclic peptide. [Item 6] The method according to Item 1, wherein the protease activity sensor is attached to a scaffold. [Item 7] The method according to Item 6, wherein the scaffold is a nanoparticle. [Item 8] The method according to Item 7, wherein multiple protease activity sensors are attached to nanoparticles. [Item 9] The method of item 1, wherein identification of the activity of the first protease and the second protease in a biological sample indicates protease dysregulation. [Item 10] The method of Item 9, wherein protease dysregulation indicates the presence of a diseased state in a biological sample. [Item 11] The method according to Item 10, wherein the disease state is cancer, fibrosis, a blood disease, an immune disease, a viral infection, or a bacterial infection. [Item 12] The method of Item 1, wherein the activity of the first protease and / or the activity of the second protease is promoted by a therapeutic agent. [Item 13] The method of item 1, wherein at least one protease is granzyme B (GzmB), thrombin (Thrb), metalloproteinase (MMP), or viral protease. [Item 14] A method for determining protease activity in a biological sample, comprising: providing a biological sample with a bicomparator comprising a reporter molecule encased in a liposome within a peptide cage, wherein cleavage of the peptide cage by a protease and perforation of the liposome by a cytolytic protein releases the reporter molecule to provide a detectable signal; detecting the presence of a detectable signal in the sample; and Identifying the activity of proteases and cytolytic proteins based on the presence of a detectable signal in the sample; The method comprising: [Item 15] The method according to Item 14, wherein the reporter molecule is a fluorescent molecule. [Item 16] The method according to Item 14, wherein the bicomparator is conjugated to the scaffold. [Item 17] The method according to Item 16, wherein the scaffold is a nanoparticle. [Item 18] The method of Item 16, wherein multiple bicomparators are attached to the scaffold. [Item 19] The method of Item 14, wherein the identification of protease and cytolytic protein activity in a biological sample indicates protease dysregulation. [Item 20] The method of Item 19, wherein protease dysregulation indicates the presence of a diseased state in a biological sample. [Item 21] The method according to Item 20, wherein the disease state is cancer, fibrosis, a blood disease, an immune disease, a viral infection, or a bacterial infection. [Item 22] The method according to Item 14, wherein the activity of the protease and / or the activity of the cytolytic protein is promoted by a therapeutic agent. [Item 23] The method according to Item 14, wherein the protease is granzyme B (GzmB), thrombin (Thrb), metalloproteinase (MMP), or viral protease. [Item 24] A composition comprising a protease activity sensor, the protease activity sensor comprising: a first protease substrate and a second protease substrate, wherein the first substrate is cleaved by the first protease and the second substrate is cleaved by the second protease, and the protease sensor provides a detectable signal when both the first substrate and the second substrate are cleaved; The composition comprising: [Item 25] The composition according to Item 24, further comprising a biological sample. [Item 26] The composition of Item 24, wherein the protease sensor is conjugated to a reporter molecule that provides a detectable signal. [Item 27] ​​The composition according to Item 26, wherein the reporter molecule is a fluorescent molecule. [Item 28] The composition of Item 27, wherein the first substrate and the second substrate are conjugated to a fluorescent quencher, and cleavage of the first substrate and the second substrate enables the fluorescent molecule to provide a detectable signal. [Item 29] The composition described in Item 24, wherein the protease sensor is a cyclic peptide. [Item 30] The composition described in Item 24, wherein the protease sensor is bound to a scaffold. [Item 31] The composition according to Item 30, wherein the scaffold is a nanoparticle. [Item 32] The composition described in Item 31, in which multiple protease sensors are bound to nanoparticles. [Item 33] The composition described in Item 24, wherein at least one of the protease substrates is cleaved by granzyme B (GzmB), thrombin (Thrb), metalloproteinase (MMP), or viral protease. [Item 34] A composition comprising a bicomparator, the bicomparator comprising a reporter molecule encapsulated in a liposome within a peptide cage, wherein cleavage of the peptide cage by a protease and perforation of the liposome by a cytolytic protein releases the reporter molecule to provide a detectable signal. [Item 35] The composition according to Item 34, further comprising a biological sample. [Item 36] The composition according to Item 34, wherein the reporter molecule is a fluorescent molecule. [Item 37] The composition described in Item 34, wherein the peptide cage is cleaved by at least one of granzyme B (GzmB), thrombin (Thrb), metalloproteinase (MMP), or viral protease. [Explanation of symbols]

[0121] 401 Bicomparator 402 Peptide Cage 403 Liposomes 404 Reporter molecules 405 Protease 606 Cytolytic Proteins 701 Method 703 Subjects 705 Administration to subjects 707 Action on protease activity sensor or biocomparator 709 Reporter Molecules 711 Collection of samples from subjects 713 Assays for detecting signals in samples 801 Cyclic Peptides 803 Cyclization Linker 805 Cyclized residue 807 spacer residues 809 Protease-specific substrates

Claims

1. 1. A non-invasive method for identifying the activity of a first protease and a second protease in a biological sample, comprising: providing a protease activity sensor to a biological sample, the protease activity sensor comprising a first protease substrate and a second protease substrate, wherein the first substrate is cleaved by the first protease and the second substrate is cleaved by the second protease, and when both the first substrate and the second substrate are cleaved, the protease activity sensor releases a reporter molecule, which provides a detectable signal, wherein the reporter molecule is released by protease cleavage and diffuses into circulation and tissues that can be non-invasively obtained; detecting the presence of a detectable signal in a non-invasively obtained sample; and identifying the activity of the first protease and the second protease based on the presence of a detectable signal in the sample; The protease activity sensor comprises the sequence of SEQ ID NO: 1 or SEQ ID NO:

2. The method.

2. The method of claim 1 , wherein the protease activity sensor is conjugated to a reporter molecule that provides a detectable signal.

3. The method of claim 2 , wherein the reporter molecule is a fluorescent molecule.

4. 4. The method of claim 3, wherein the first substrate and the second substrate are conjugated to a fluorescent quencher, and cleavage of the first substrate and the second substrate enables the fluorescent molecule to provide a detectable signal.

5. The method of claim 1 , wherein the protease activity sensor is a cyclic peptide.

6. The method of claim 1 , wherein the protease activity sensor is attached to a scaffold.

7. The method of claim 6 , wherein the scaffold is a nanoparticle.

8. The method of claim 7 , wherein a plurality of protease activity sensors are attached to a nanoparticle.

9. 10. The method of claim 1, wherein identification of the activity of the first protease and the second protease in the biological sample indicates the presence of a diseased state in the biological sample.

10. 10. The method of claim 9, wherein the disease state is cancer, fibrosis, a blood disorder, an immune disorder, a viral infection, or a bacterial infection.

11. 10. The method of claim 1, wherein the activity of the first protease and / or the activity of the second protease is stimulated by a therapeutic agent.

12. 2. The method of claim 1, wherein the at least one protease is granzyme B (GzmB), thrombin (Thrb), a metalloproteinase (MMP), or a viral protease.

13. 1. A composition comprising a protease activity sensor, the protease activity sensor comprising: a first protease substrate and a second protease substrate, wherein the first substrate is cleaved by the first protease and the second substrate is cleaved by the second protease, and when both the first and second substrates are cleaved, the protease activity sensor releases a reporter molecule, which reporter molecule provides a detectable signal; Including, The protease activity sensor comprises the sequence of SEQ ID NO: 1 or SEQ ID NO:

2. The composition.

14. The composition of claim 13 , further comprising a biological sample.

15. 15. The composition of claim 14, wherein the protease activity sensor is conjugated to a reporter molecule that provides a detectable signal.

16. The composition of claim 15 , wherein the reporter molecule is a fluorescent molecule.

17. 17. The composition of claim 16, wherein the first substrate and the second substrate are conjugated to a fluorescent quencher, such that cleavage of the first substrate and the second substrate enables the fluorescent molecule to provide a detectable signal.

18. The composition of claim 14 , wherein the protease activity sensor is a cyclic peptide.

19. The composition of claim 14 , wherein the protease activity sensor is attached to a scaffold.

20. 20. The composition of claim 19, wherein the scaffold is a nanoparticle.

21. 21. The composition of claim 20, wherein a plurality of protease activity sensors are attached to the nanoparticles.

22. 15. The composition of claim 14, wherein at least one of the protease substrates is cleaved by granzyme B (GzmB), thrombin (Thrb), a metalloproteinase (MMP), or a viral protease.

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