Compositions and methods for bioactive logic gating analysis

Abstract

The present disclosure provides methods and compositions for digital analysis of biological activity by detecting the activity of at least two biomarkers using protease activity sensors or biological comparators.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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.

[0003] GOVERNMENT FUNDING ADMISSION

[0004] This application was funded by the National Institutes of Health under grant number DP2HD091793. The government has certain rights in the application. TECHNICAL FIELD

[0005] The present disclosure relates to tools, compositions, and methods for digitizing biological activity. BACKGROUND

[0006] Advances in human health depend on improving the ability to extract meaningful information from complex biological states and using this information to guide diagnostic, prognostic, or therapeutic interventions. Currently, clinical decisions rely on extracting information from complex biological states by demodulating “analog” signals, such as biomarker levels, which are prone to noise, imprecision, and heterogeneity. Applying computational principles to biological systems can improve the accuracy and sensitivity of extracting information from complex biological states, enabling accurate disease diagnosis and increasing medical efficacy. For example, computation uses “digital” signals that can robustly transmit information and are able to tolerate uncontrollable variables. Using compositions and methods that enable analysis of biological activity under a digital framework, such as Boolean logic, can significantly improve the ability to extract meaningful information from complex biological states.

[0007] The enzymatic activity of certain proteases has been shown to play a vital role in monitoring and diagnosing certain pathologies and treatments. Over 550 proteases are encoded by the human genome, and dysregulation of the protease signaling network is the biological basis of many pathologies, including cancer, fibrosis, hematological, 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 developing therapeutic means. This is especially true for cancer, where proteases play a critical role in directing tumorigenesis.

[0008] Accurate detection of protease activity can also provide information on the effectiveness and specificity of particular therapeutic means. One important emerging class of therapeutic means is immunotherapy. Immunotherapy harnesses the immune system to treat a variety of diseases, such as cancer, organ transplant rejection, infectious diseases, allergic diseases, autoimmunity, and chronic inflammation. These therapies employ therapeutic antibodies, cytokines, and cell-based therapies, while harnessing both the humoral and cellular arms of the immune response. Despite the broad potential of immunotherapy, many patients fail to exhibit clinical benefit, while others can develop immunotherapy resistance. Patients who respond to immunotherapy often exhibit an atypical response pattern that can be misinterpreted as disease progression. Due to insufficient technology for response monitoring and for determining potential resistance mechanisms, not only does disease persist in the population, but drug development and clinical trials also face significant obstacles. In order to realize full benefit from immunotherapy, methods for monitoring biomarkers, including protease activity, during immunotherapy need to be improved.

[0009] Existing methods for measuring protease activity, such as structural biology, enzymology, and inhibitor-based assays, have provided important information about proteases. However, these techniques are often lacking in sensitivity and are unable to distinguish between various disease states or off-target activity.

[0010] For diagnosis, tissue biopsy remains the gold standard for diagnosis, but it is invasive and samples less than 0.1% of the total disease site. (Cyll et al., Br J Cancer, 117(3): 367-375 (2017)). Liquid biopsy offers a non-invasive means, but dilution of biomarkers in blood significantly limits sensitivity ((Nagrath, S., et al., Nature, 450(7173): 1235-1239 (2007); Hori, et al., Sci Transl Med, 3(109): 109ral6 (2011)). Imaging techniques can also be limited by low sensitivity and specificity, as well as the atypical response patterns often associated with immunotherapy, which can lead to misclassification of responsive patients as cases of treatment failure. Development of better non-invasive biomarkers will more quickly identify responsive patients and elucidate mechanisms of new immunotherapies.

[0011] Accordingly, it is an object of the present invention to provide a bioactivity sensor that utilizes protease biology for specific and sensitive non-invasive diagnosis. SUMMARY

[0012] The present disclosure provides methods and compositions for the digital analysis of biological activity by detecting the activity of at least two biomarkers. The biomarkers can be proteins or enzymes, including proteases and / or cytolytic proteins. The methods and compositions use a digital framework to detect the activity of the biomarkers. To establish the digital framework, the present disclosure provides a logic-gated biomarker activity sensor. The activity sensor uses AND-gated logic to probe the activity of two biomarkers. The activity sensor senses biological activity as bits by specific biomarkers. Activity of one biomarker represents a state of "1", while lack of activity represents a state of "0". Activity of both biomarkers represents a state of "1,1" and provides a TRUE output. Activity of only one of the two biomarkers represents a state of "1,0", while lack of biomarker activity represents a state of "0,0". Both states of "0,1" and "0,0" provide a FALSE output.

[0013] The activity sensor can comprise protease substrates designed to sense biological activity as biological bits by specific protease cleavage events, where uncleaved substrate represents a state of "0" and cleaved substrate represents a state of "1". The sensor uses AND-gated logic to simultaneously probe the activity of two proteases, providing a TRUE output only after cleavage events occur under the action of both proteases. Cleavage of one protease substrate or lack of substrate cleavage provides a FALSE output. Using this concept, the present disclosure provides methods and compositions using logic-gated activity sensors that utilize protease activity to provide, for example, specificity and sensitivity for non-invasive diagnostics.

[0014] The present disclosure provides methods and compositions for determining protease activity in a biological sample using a protease activity sensor. The protease activity sensor comprises a first protease substrate and a second protease substrate. Each substrate is cleaved by a different protease, e.g., a first protease and a second protease. The sensor can employ AND-gated logic. Thus, when both substrates are cleaved, the protease activity sensor provides a detectable signal (a TRUE output) indicating activity of the first and second proteases in the sample. However, if only one or no substrate is cleaved, no detectable signal is produced (a FALSE output).

[0015] The protease sensor can be conjugated to a reporter molecule, such as a fluorescent molecule, to provide a detectable signal. When the reporter molecule is a fluorescent molecule, the first and second protease substrates can be conjugated to a fluorescence quencher. Cleavage of the protease substrates removes the quencher, which enables the fluorescent molecule to provide a detectable signal (a TRUE output). However, if one or both protease substrates are not cleaved, the fluorescence quencher is not removed and prevents a detectable signal (a FALSE output).

[0016] The protease sensor can be a cyclic peptide. The cyclic peptide can comprise two different protease substrates. The substrates can separate a fluorescent reporter molecule from a quencher.

[0017] The protease sensor can be conjugated to a scaffold, such as a nanoparticle. Multiple protease sensors can be conjugated to the scaffold. Conjugating the protease sensors to the scaffold can increase their presentation valency, increasing the rate of proteolysis and thereby amplifying the detectable signal. This can improve the signal-to-noise ratio, especially when the protease sensors are delivered in vivo.

[0018] The present disclosure provides methods and compositions for determining biomarker activity in a biological sample using a dual comparator. The dual comparator can comprise a reporter molecule encapsulated in a liposome. The liposome is housed 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, thereby releasing the reporter molecule. The dual comparator can employ AND-gate logic. Upon release, the reporter molecule provides a detectable signal (a true output), which indicates the activity of the protease and cytolytic protein in the sample. If there is no protease capable of cleaving the peptide cage and no cytolytic protein to perforate the liposome, the reporter molecule is not released and no detectable signal is produced (a false output). The reporter molecule can be a fluorescent molecule.

[0019] The dual comparator can be conjugated to a scaffold, such as a nanoparticle. Multiple dual comparators can be conjugated to the scaffold. Conjugating the multiple dual comparators to the scaffold can increase their presentation valency, increasing the rate of proteolysis and thereby amplifying the detectable signal. This can improve the signal-to-noise ratio, especially when the dual comparators are delivered in vivo.

[0020] The protease substrates, including the peptide cage of the dual comparator, are designed to be cleaved by a specific protease. This allows for the determination of the presence of the specific protease in the sample. The presence or absence of the specific protease can be indicative of protease dysregulation. Protease dysregulation can be indicative of the presence of a disease state, such as cancer, fibrosis, a hematological disorder, an immune disorder, a viral infection, or a bacterial infection. Thus, the methods and compositions of the present disclosure can be used to diagnose a disease or progression of a condition.

[0021] Protease activity can be indicative of an immune response to an immunotherapy or therapeutic drug. Thus, the methods and compositions of the present disclosure can be used to monitor immunotherapy and therapeutic drug treatments. Monitoring can include measuring the efficacy, specificity, and response of a particular treatment. This ability of the disclosed methods and compositions is particularly useful for cancer immunotherapy.

[0022] Monitoring the immune response can include detecting the activity of one or more proteases and / or cytolytic proteins. The activity of the one or more proteases and / or cytolytic proteins can be promoted by the therapeutic agent. In this way, the activity, specificity, and / or efficacy of the therapeutic agent can be monitored.

[0023] Cancer immunotherapy is limited by its off-target effects. Non-invasive methods of detecting immune activity within a tumor greatly improve treatment monitoring. Monitoring a patient’s immune activity alone can not confirm the efficacy of an immunotherapy because, for example, the immune system is activated against another pathology (e.g., viral infection). The specificity provided by the AND-gated protease activity sensor and the dual comparator enables, for example, distinguishing between immune activity in a tumor and immune activity caused by a viral infection. The methods and compositions of the present disclosure enable monitoring immune activity within a tumor and can distinguish between immune activity caused by a tumor, a therapeutic means, and another pathology such as a viral infection.

[0024] The methods and compositions of the present disclosure can be used in vitro or in vivo. The methods of the present disclosure can include administering to a subject an amount of a protease sensor or dual comparator as described herein. The method 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 be indicative of protease dysregulation and / or an immune response in the subject. The protease dysregulation can be indicative of a disease state in the subject. The disease state can be a cancer, a fibrosis, a hematological 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 method can include monitoring the activity of at least one of granzyme B (GzmB), thrombin (Thrb), a metalloproteinase (MMP), or a viral protease. BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1 The principles behind digitizing the readout of biological activity are shown.

[0026] FIG. 2 A schematic of an example of a protease activity sensor is shown.

[0027] FIG. 3 The principles behind the AND-gated logic in a protease activity sensor are shown.

[0028] FIG. 4 A schematic of an AND-gated biological comparator is shown.

[0029] FIG. 5 A schematic of a biological comparator with a cleaved peptide cage is shown.

[0030] FIG. 6 A schematic of a biological comparator with a perforated liposome and released reporter molecule is shown.

[0031] FIG. 7 An exemplary method of the present disclosure is shown.

[0032] FIG. 8 An exemplary circular protease activity sensor is shown.

[0033] FIG. 9 A schematic of an exemplary protease activity sensor is shown.

[0034] FIGS. 10-14 Experimental results for an exemplary protease activity sensor are shown.

[0035] FIG. 15 A schematic of a protease activity sensor is shown.

[0036] FIGS. 16-17 Experimental results for a protease activity sensor are shown.

[0037] FIG. 18 A schematic of a protease activity sensor is shown.

[0038] FIGS. 19-21 Experimental results for a protease activity sensor are shown.

[0039] FIGS. 22-23 Experimental results related to a biological comparator are shown.

[0040] FIG. 24 A schematic of an experimental protocol using a biological comparator is shown.

[0041] FIG. 25 A schematic and experimental results for a biological comparator are shown. DETAILED DESCRIPTION

[0042] The present disclosure provides methods and compositions for digital analysis of biological activity by detecting the activity of at least two biomarkers. The biomarkers can include proteins or enzymes, including proteases and / or cytolytic proteins. The methods and compositions use a digital framework to detect the activity of the biomarkers. 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 as bits through specific biomarkers. Activity of one biomarker represents a state of "1", and lack of activity represents a state of "0". Activity of both biomarkers represents a state of "1,1" and provides a true value output. Activity of only one of the two biomarkers represents a state of "1,0" and lack of activity of either biomarker represents a state of "0,0". States "0,1" and "0,0" both provide a false value output. Since the methods and compositions require activity of two specific biomarkers, they can reliably provide digital analysis of biological activity.

[0043] Enzymes are differentially expressed under different physiological states of interest, such as in response to disease, infection, immune response, or therapeutic means. For example, dysregulation of protease activity can be indicative of a disease state. Dysregulated proteases have important influences in disease, including cancer, progression as they can alter cell signaling, helping to drive cancer proliferation, invasion, angiogenesis, evasion of apoptosis, and metastasis. However, the activity of a single enzyme, including proteases, is often a determinant factor of a biological response. By detecting the activity of multiple enzymes, such as proteases and cytolytic proteins, a binary signal, such as a true or false output, can be used to more reliably determine and report the response.

[0044] The methods and compositions can be used to monitor and diagnose disease, immune response, and therapeutic effects. Monitoring can include longitudinal monitoring, which includes, for example, changes in disease state, immune activity, and / or response to therapeutic effects. The methods and compositions can be used for longitudinal monitoring and personalized medicine, as well as for the recruitment, enrichment, qualification, and stratification of study participants. The methods and compositions can be used to detect therapeutic efficacy, monitor response to therapy over time, and detect relapse and remission. Detection of reporter molecules in a sample from a subject can be indicative of the presence of a disease in the subject, the stage of the disease, and the rate or level of disease activity. Testing using the compositions of the present disclosure can be administered and read non-invasively, quickly, and without the need for imaging by X-ray or otherwise.

[0045] The compositions of the present disclosure can be used to determine the responsiveness and therapeutic effectiveness of a subject to a drug. For example, the compositions can be administered and tested at multiple time points and read to observe trends in disease change or progression or remission over time. The compositions can be used to measure activity in vivo, whether or not the subject has received a treatment or the compositions are used for diagnosis. Based on this, the present disclosure provides additional understanding of the disease, rather than just the effect on the disease. The compositions can be used to study the disease and / or other related activities. For example, the activity or progression of the disease can be detected, such as the rate of growth of a tumor. For subjects receiving a drug treatment, the compositions can be used to detect activity indicative of whether the participant is responding to the treatment. For example, the activity sensor can be designed to detect whether the size of a tumor is growing or shrinking, the rate of activity or progression of the disease, whether a particular dosage of a drug is effective, and whether the participant is likely to respond to the treatment.

[0046] This method and composition can be used to detect the activity of two specific biomarkers to digitally analyze whether a subject responds to multiple treatments. By measuring the activity of two specific biomarkers, the methods and compositions of this disclosure enable a more accurate determination of the type of response in a subject using binary signals. For example, certain drugs are used to treat diseases. However, the drug may involve different approaches to treating the disease. In treating diseases such as non-alcoholic steatohepatitis (NASH), one drug may be an antifibrotic drug, while another may be an anti-inflammatory or anti-NASH drug. Detecting the activity of two drugs by demodulating the activity of biomarkers associated with each drug invoked in the participant's body helps to gain deeper insights into why a participant responds to or does not respond to treatment.

[0047] Furthermore, this method and composition can be used to detect whether a subject will respond to treatment. For example, a subject may have a tumor that has been treated or is currently being treated with a checkpoint blockade drug. The subject may appear unresponsive to treatment because the tumor size continues to grow after checkpoint blockade is administered. However, because this invention detects the bioactivity of two biomarkers in the body, it can detect whether this growth is due to an influx of immune cells. Simultaneously, due to the specificity imparted by the methods and compositions of this disclosure, it is possible to distinguish whether the immune response is caused by the checkpoint blockade drug or by a lesion (such as a viral infection). Therefore, it is determined that the subject will respond to treatment, rather than being unresponsive.

[0048] The methods and compositions disclosed herein may include a protease activity sensor. This activity sensor may include multiple reporter molecules that are detectable in a sample, but only when two proteases come into contact and cleave, and are associated with local immune responses or cancer progression. Cleavage of only one protease will not result in a state represented by "1,1" and will therefore provide a false output. Cleavage of both proteases will result in a state represented by "1,1" and will therefore provide a true output. The protease sensor may be provided to a subject in vivo, and the reporter molecules will be detectable in bodily fluids only after cleavage of the protease that releases the reporter molecules.

[0049] like FIG. 1 As shown in Figure A, the protease activity sensor disclosed in this subject matter enables the provision of biological activity in digital readings. As described herein, the protease activity sensor can provide non-invasive reporting of the activities of two proteases by designing two protease-specific cleavage sites on the sensor. For example, a protease-specific substrate (e.g., a cleavage site) can be cleaved by a protease that is promoted by cancerous or immune activity.

[0050] FIG. 2 A non-limiting schematic diagram of a protease activity sensor is provided. (e.g.) FIG. 2As shown, a plurality of protease activity sensors can be attached to a scaffold, such as a nanoparticle. The protease activity sensors each have a reporter molecule, such as FIG. 2 As shown, the reporter molecule can be a fluorescent molecule. The reporter molecule is attached to two different protease substrates, which 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 fluorescence quencher, such as FIG. 2 As shown in the black circle in FIG. 2 As shown, the release can allow the reporter molecule to move away from the fluorescence quencher, allowing a detectable signal to appear.

[0051] FIG. 3 The protease activity sensors are shown as providing AND-gated logic by providing a true value output only after cleavage events by both proteases, to simultaneously probe the activity of two proteases. The “0,0” signal results from no protease cleavage and provides a false value output. The “0,1” signal results from protease cleavage by only one protease and provides a false value output. The “1,1” signal results from protease cleavage by both proteases and provides a true value output.

[0052] The methods and compositions of the present disclosure can include a dual comparator. The dual comparator can include a plurality of reporter molecules. The reporter molecules are encapsulated in liposomes. The liposomes are housed within a peptide cage, which is a substrate for a particular protease. Cleavage of the peptide cage by the protease releases the liposomes. The liposomes can be perforated by a cytolytic protein, such as perforin. The reporter molecules can only be detected if the peptide cage is cleaved by the protease and the liposomes are perforated by the cytolytic protein. Release of the reporter molecules results in a state represented by “1,1” and thus will provide a true value output. The dual comparator can be provided to a subject in vivo and the reporter molecules can only be detected in a bodily fluid after release of the reporter molecules.

[0053] FIGS. 4-6 A non-limiting schematic of a dual comparator is provided. As shown in FIG. 4 The dual comparator 401 has a reporter molecule 404, as shown by the star. The reporter molecule can be a fluorescent molecule. The reporter molecule 404 is encapsulated within a liposome 403. The liposome 403 is housed within a peptide cage 402. A protease 405 contacts the dual comparator 401. As shown in FIG. 5 The protease cleaves the peptide cage. Then, as shown in FIG. 6 A cytolytic protein 606 (e.g., perforin) perforates the liposome and releases the reporter molecule, providing a detectable signal.

[0054] The present disclosure includes methods of administering a protease activity sensor or dual comparator of the present disclosure to a subject in vivo to monitor or diagnose a disease or treatment efficacy. FIG. 7 A non-limiting example of this method 701 is shown. The method 701 can be implemented in the context of a longitudinal study to determine the efficacy of a treatment modality. The subject 703 includes a subject who is about to receive or is receiving a treatment. A protease activity sensor or dual comparator is administered 705 to the subject. The method 701 can include monitoring changes in the disease status of a subject who has received a disease treatment modality by measuring at multiple points over time (i.e., in a longitudinal manner). Two specific biomarkers contact the protease activity sensor or dual comparator, which act on the protease activity sensor or dual comparator 707. This causes the protease activity sensor or dual comparator to release a reporter molecule 709. A sample is obtained 711 from the subject, and an assay is performed to detect the signal in the sample 713. By detecting the reporter molecule in the sample from the subject, the activity of the two biomarkers in the sample is determined, and thus their presence is determined. The presence and activity of the two biomarkers in the sample can indicate that the treatment modality has promoted progression of the disease state or an immune response.

[0055] The method 701 can be used to detect disease-related activity in vivo, such that the activity of the two biomarkers detected using the sensor indicates disease progression or treatment efficacy. The protease activity sensor or dual comparator can be administered over time to monitor the subject’s response to a treatment and show whether the treatment is effective to treat the disease in the subject. Different drugs or combinations of drugs can be studied in different subjects or at different times to determine drugs or combinations that are effective to treat a condition. The protease activity sensor or dual comparator provides a marker of health / disease progression and provides this marker very rapidly (the signal in a sample from the subject can be detected within hours of administering the protease activity sensor or dual comparator). The protease activity sensor or dual comparator can specifically report the activity of multiple enzymes, including some enzymes that are dysregulated in a disease state and others that are specific to a comorbidity. Preferably, the expression of the enzymes (e.g., extracellular proteases) is upregulated due to a certain pathology, treatment, or condition.

[0056] The method 701 can be used to monitor cancer progression in a subject. The subject can be suspected of having cancer, known to have cancer (in an active or remission phase), at risk of having cancer, and / or undergoing a cancer treatment (including an immuno-oncology (I-O) therapy). The protease sensor can be administered 705 to the subject. The sensor can include a reporter molecule linked by two protease substrates. The protease substrates are each sensitive to a protease whose activity is indicative of a feature in the tumor environment (e.g., an enzyme that is upregulated in tumor expansion or tumor regression, or an enzyme that is indicative of an immune response being active or suppressed).

[0057] Similarly, a biological comparator can be administered 705 to the subject. The biological comparator can have a peptide cage that is a substrate for a protease, whose activity is indicative of a feature in the tumor environment. The liposome of the biological comparator can be perforated by a protein, whose activity is indicative of a feature in the tumor environment.

[0058] As described herein, the activity sensors are designed to report on the disease and treatment status of a patient, according to protease activity, information obtained from the level of reporter molecules in a patient sample can be used to diagnose and / or stage the disease, monitor progression, predict responsiveness to a given therapy, and monitor treatment effectiveness, including distinguishing between anti-tumor immune responses, systemic immune responses, and tumor progression. The activity sensors can be administered by any suitable method. The activity sensors can be delivered intravenously or nebulized and delivered to the lungs, e.g., via a nebulizer. In other examples, the activity sensors can be administered to a subject transdermally, intradermally, intra-arterially, intra-lesionally, intra-tumorally, intracranially, intra-articularly, intra-tumorally, intramuscularly, subcutaneously, orally, topically, locally, by inhalation, by injection, by infusion, or by other methods known in the art or any combination thereof.

[0059] Proteases are a class of enzymes, including over 550 members encoded in the human genome, many of which have disease-specific roles, including key roles in immunity. For example, cytotoxic T cell-mediated target cell killing is a protease-driven process, including: 1) death receptor signaling and caspase (a protease whose activity can mediate cell death) activation, and 2) secretion of granzymes (proteases that enter target cells through a perforin-dependent mechanism to activate caspase-mediated cell death). In addition, proteases are critical 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, are established hallmarks of cancer (Arias et al., Trends Cancer, 3(6):407-422 (2017); Egeblad et al., Nat Rev Cancer, 2(3): 161-174 (2002)).

[0060] Given the central role proteases play in the underlying biology of immunity, oncology, and pathophysiology of various diseases, proteases provide an innovative means for immune therapy response monitoring (Dudani et al., Ann Rev of Cancer Biology, (2018)). For example, a hallmark of “hot” tumors is effective immune infiltration by cytotoxic T cells that kill cancer cells primarily through a perforin-dependent, granzyme-mediated pathway, the latter of which comprises a potent family of serine proteases (Larimer et al., Cancer Res, 77(9):2318-2327 (2017); Voskoboinik et al., Nat Rev Immunol, 15(6):388-400 (2015)). Expression of proteases, including inflammatory and matrix-degrading proteases, in tumors has been recognized as a hallmark of underlying 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 indications of drug response. Therapeutic early immune and disease site-specific protease activity enables identification of active biomarkers that are predictive of therapeutic efficacy and indicative of resistance to immunotherapy.

[0061] In one embodiment, the protease amplifies the detection signal at the disease or treatment site (x 1000 fold). Upon protease cleavage, the reporter molecule is concentrated into the urine, rather than diluted in the blood, further enriching the signal by 100 fold. This enables, for example, ultrasensitive early detection of T cell activity before radiological imaging of the disease site can detect changes.

[0062] Protease substrates comprise a recognition sequence that is cleavable by a protease. Cleavage of the protease substrate can release a reporter molecule attached to the substrate. The protease substrates can each be a substrate for a specific protease known to be associated with a diseased cell. Proteases known to be associated with a diseased cell or tissue include, but are not limited to, serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide cleaving enzymes, serum proteases, cathepsins, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hKl, hKlO, hKl 5, fibrinolysin, collagenase, collagenase type IV, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidain, bromelain, calpain, caspases, caspase-3, Mir 1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMPs), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase-type plasminogen activator (uPA), enteropeptidase, prostate specific antigen (PSA, hK3), interleukin-1 beta converting enzyme, thrombin, FAP (FAP-alpha), dipeptidyl peptidases, meprins, granzymes, and dipeptidyl peptidase IV (DPPIV / CD26).

[0063] The protease substrates can be tumor-specific protease substrates. Exemplary 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 15, uPA, uPAR, caspases, matrix metalloproteases such as MMP1, MMP2, MMP8, MMP9, MMP13, MMP14, and ADAM. In another embodiment, the protease substrate is a cell-specific protease substrate, such as a T cell-specific protease substrate. Exemplary 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 proteases of the caspase family such as caspase-3, -6, -7, -8.

[0064] The protease substrate can be one specific to a protease associated with inflammation and / or programmed cell death. Several proteases are known to be associated with inflammation and programmed cell death (e.g., including apoptosis, pyroptosis, and programmed necrosis). Accordingly, the level of activity of these proteases is indicative of immune system activity. Caspases (cysteine-aspartate proteases, cysteine aspartases, or cysteine-dependent aspartate-directed proteases) are a family of proteases that contain a cysteine in their active site that only cleaves target proteins after an aspartate residue nucleophilically. Caspase-1, caspase-4, caspase-5, and caspase-11 are associated with inflammation. Serine proteases also play a role in apoptosis and inflammation, so their differential expression is also indicative of an immune response. Immune cells express serine proteases, such as granzymes, neutrophil elastase, cathepsin G, proteinase 3, chymase, and tryptase.

[0065] The disclosed protease activity sensors and biological comparators can be used to distinguish between programmed cell death indicative of an immune response and necrosis that occurs naturally during tumor progression. Unlike programmed cell death, where caspases and serine proteases are the primary proteases, calpains and lysosomal proteases (e.g., cathepsins B and D) are the key proteases of necrosis. Thus, the levels of calpain and cathepsin indicated by the activity sensor reporter molecule measurements can provide information about necrotic cell death to complement the immuno-oncology information.

[0066] The protease activity sensor can release a reporter molecule after cleavage by two different proteases. The detectable signal can be the cleavage product of the protease substrate itself or a peptide fragment. Upon cleavage, the fragment of the protease substrate is released into the circulation and detected in the urine by mass spectrometry. Cleavage by one protease can release the reporter molecule. However, the detectable signal cannot be detected from the released reporter molecule until cleavage by a second protease. For example, such second cleavage can cleave a fluorescence quencher off of the reporter molecule, allowing detection. Alternatively, the protease substrate is designed with a quencher molecule before the cleavage site and a fluorescent reporter after the cleavage site. 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 the urine by standard methods such as flow cytometry.

[0067] The protease substrate can be conjugated to a reporter molecule, quencher, and / or scaffold using methods known in the art. In one embodiment, the protease substrate can be conjugated by introducing a linker between the protease substrate and the reporter molecule, quencher, and / or scaffold that forms a covalent conjugate. Exemplary 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.

[0068] The reporter molecule released from the active sensor of the present application can be detected by any suitable detection method capable of directly or indirectly detecting the presence of the reporter molecule. For example, the reporter molecule can be detected by a ligand binding assay, which is a test that involves the binding of a capture ligand to an affinity agent. After capture, the reporter molecule can be directly detected by optical density, radioemission, or non-radiative energy transfer. Alternatively, the reporter molecule can be indirectly detected using an antibody conjugate, an affinity column, a streptavidin-biotin conjugate, a PCR analysis, a DNA microarray, or a fluorescence analysis.

[0069] The ligand binding assay typically involves a detection step such as an ELISA (including fluorescent, colorimetric, bioluminescent, and chemiluminescent ELISA), a paper test strip, or a lateral flow assay, or a microbead-based fluorescence assay.

[0070] In one example, a paper-based ELISA test can be used to detect the reporter molecule released in urine. Paper-based ELISAs can be created inexpensively, such as by reflowing the wax deposited from a commercial solid inkjet printer to create a series of test points on a single sheet of paper. When the solid ink is heated to a liquid or semi-liquid state, the printed wax penetrates into the paper, forming a hydrophobic barrier. The spaces between the hydrophobic barriers can then be used as individual reaction wells. The ELISA test can be performed by drying detection antibodies on the individual reaction wells, followed by blocking and washing steps to set up the test points on the paper. Urine from a urine sample taken from a subject can then be added to the test points, and subsequently a streptavidin alkaline phosphatase (ALP) conjugate can be added to the test points as the detection antibody. Next, the bound ALP can be exposed to a chromogenic reagent, such as BCIP / NBT (5-bromo-4-chloro-3'-indolyl phosphate p-toluidine salt / nitro blue tetrazolium chloride), which results in a purple precipitate indicating the presence of the reporter molecule.

[0071] In another example, volatile organic compounds can be detected by an analytical platform such as a gas chromatograph, a breathalyzer, a mass spectrometer, or using optical or acoustic sensors.

[0072] Gas chromatography can be used to detect compounds that do not decompose and can be vaporized (e.g., volatile organic compounds). Gas chromatographs include a mobile phase (or mobile phase) which is a carrier gas (e.g., an inert gas such as helium and an inactive gas such as nitrogen) and a stationary phase which is a microscopic layer of liquid or polymer on an inert solid support, located within 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, resulting in their elution at different times (i.e., different retention times in the column). Compounds can be distinguished by their retention times.

[0073] Improved breathalyzers can also be used to detect volatile organic compounds. In a traditional breathalyzer used to detect alcohol levels in the blood, the subject breathes into the instrument, and any ethanol present in the subject’s breath is oxidized to acetic acid at an anode. At a cathode, oxygen from the atmosphere is reduced. The overall reaction is the oxidation of ethanol to acetic acid and water, which produces a current that can be detected and quantified by a microcontroller. Improved breathalyzers that utilize other reactions can be used to detect a variety of volatile organic compounds.

[0074] Mass spectrometry can be used to detect and distinguish reporter molecules based on mass differences. In mass spectrometry, a sample is ionized, for example by bombarding it with electrons. The sample can be a solid, liquid, or gas. By ionizing the sample, some of the molecules of the sample are fragmented into charged fragments. These ions can then be separated according to their mass-to-charge ratio. This is typically done by accelerating the ions and placing them in an electric or magnetic field, in which ions with the same mass-to-charge ratio will experience the same amount of deflection. When deflected, the ions can be detected by a mechanism capable of detecting charged particles, such as an electron multiplier. The results of the detection can be displayed as a spectrum of the relative abundance of detected ions as a function of mass-to-charge ratio. Molecules in the sample can then be identified by correlating known masses (such as the mass of the entire molecule) with the identified masses or by characteristic fragmentation patterns.

[0075] When the reporter molecule comprises a nucleic acid, the reporter molecule can be detected by various sequencing methods known in the art, such as traditional Sanger sequencing or by next generation sequencing (NGS) technology. NGS generally refers to non-Sanger-based high-throughput nucleic acid sequencing technologies in which many (i.e., thousands, millions, or billions) of nucleic acid strands can be sequenced in parallel. Examples of such NGS sequencing include platforms produced by Illumina (e.g., HiSeq, MiSeq, NextSeq, MiniSeq, and iSeq100), Pacific Biosciences (e.g., Sequel and RSII), and Ion Torrent under ThermoFisher (e.g., Ion S5, Ion Proton, Ion PGM, and Ion Chef systems). It will be appreciated that any suitable NGS sequencing platform can be used for NGS to detect nucleic acid of a reporter molecule as described herein.

[0076] The analysis can be performed directly on the biological sample, or the reporter molecule can be purified to some extent first. For example, a purification step can include separating the reporter molecule from other components in the biological sample. Purification can include methods such as affinity chromatography. The separated or purified reporter molecule need not be 100% pure, or even substantially pure, prior to analysis.

[0077] The reporter molecule can be attached to a label or can itself comprise a label. Suitable labels for the reporter molecule include any type of label that can be detected by standard methods, including spectroscopic, photochemical, biochemical, electrical, optical, or chemical methods. The label can be a fluorescent label. A fluorescent label is a compound that comprises at least one fluorophore. Commercially available fluorescent labels include, for example, fluorescein phosphoramidites, rhodamine, polymethadine dye derivatives, phosphors, Texas Red, green fluorescent protein, CY3, and CY5. Other known techniques, such as chemiluminescence or colorimetry (enzymatic color development reactions), can also be used to detect the reporter molecule. Quencher compositions can also be used, in which a “donor” fluorophore is linked to a “acceptor” chromophore by a short bridge that is the binding site for the enzyme. The signal of the donor fluorophore is quenched by the acceptor chromophore through a process that is 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, relief of quenching, and subsequent signal generation measured from the donor fluorophore. Examples of FRET pairs include 5-carboxyfluorescein (5-FAM) and CPQ2, FAM and DABCYL, Cy5 and QSY21, Cy3 and QSY7.

[0078] The reporter molecule can comprise one or more protease substrates designed to carry a quencher molecule before the cleavage site and a fluorophore or fluorescent reporter molecule after the cleavage site. Quencher molecules are known in the art. Exemplary quencher molecules include, but are not limited to, Deep Dark Quenchers (Eurogentec), DABCYL, TAMRA, ECLIPSE, Iowa quencher and QSY. Exemplary fluorophores or fluorescent reporter molecules include, but are not limited to, 6-FAM TM , TET TM , JOE TM , HEX TM , 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.

[0079] The protease activity sensor or biological comparator can comprise other reporter molecules such as avidin, biotin, beta-galactosidase, luciferase, alkaline phosphatase (ALP), 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 dual comparator and released into the circulation. The reporter molecule is then detected in the urine sample using an appropriate detection method such as, but not limited to, ELISA, immunoblotting, immunoassay, and bioluminescent assay.

[0080] The protease activity sensor or biological comparator can comprise a ligand to help them target specific tissues or organs. When administered to a subject, the protease activity sensor or biological comparator can be transported in the body by a variety of routes (depending on how they enter the body). For example, if administered intravenously, they will enter the systemic circulation from the point of injection and can be passively transported throughout the body.

[0081] The protease activity sensor or dual comparator can comprise a scaffold. The scaffold can further comprise or be conjugated to an adjustment domain. The adjustment domain alters the distribution or residence time of the protease activity sensor or dual comparator in the subject when administered to the subject. The protease activity sensor or dual comparator can be adjusted in a variety of ways via the adjustment domain to facilitate detection of enzyme activity in a particular cell or in a particular tissue in vivo. For example, the protease activity sensor or dual comparator can be adjusted to promote distribution to a particular tissue or to improve residence time in the subject or in a particular tissue. The adjustment domain can include, for example, molecules positioned in rapidly replicating cells to better target tumor tissue.

[0082] The protease activity sensor or dual comparator, when administered to a subject, is delivered systemically and can diffuse from the systemic circulation to a particular tissue where a reporter molecule can be released by an enzyme indicative of cancer progression or an immune response. The reporter molecule can then diffuse back into the circulation where it can be filtered by the kidneys and excreted into the urine, whereby detection of the reporter molecule in a urine sample is indicative of enzyme activity in the target tissue.

[0083] The scaffold can be any suitable platform for delivering the protease activity sensor or dual comparator systemically to a subject. The scaffold can be any material or size suitable for use as a scaffold or platform. Preferably, the scaffold is biocompatible, non-toxic, and non-immunogenic and does not elicit an immune response in the subject to which it is administered. The scaffold can also serve as a targeting device to target the protease activity sensor or dual comparator to a tissue, cell, or molecule. The scaffold can be a polymeric scaffold. For example, the scaffold can cause passive targeting of a tumor or other particular tissue by the circulation. Other types of scaffolds include, for example, compounds that facilitate active targeting of a tissue, cell, or molecule. 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.

[0084] The scaffold can include a variety of materials, such as iron, ceramic, metal, natural polymeric materials (such as hyaluronic acid), synthetic polymeric materials (such as polyglycolsuccinate), and non-polymeric materials or combinations thereof. The scaffold can be composed entirely or in part 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 acrylates and methacrylates, methyl cellulose, ethyl cellulose, and hydroxypropyl cellulose. Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

[0085] Examples of biodegradable polymers include synthetic polymers (such as polymers of lactic and glycolic acids, poly-anhydrides, polyurethanes) and natural polymers (such as alginate and other polysaccharides including dextran and cellulose, collagen, albumin and other proteins), copolymers and mixtures thereof. Generally, these biodegradable polymers degrade by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. These biodegradable polymers can be used alone, as physical mixtures (blends) or as copolymers.

[0086] In preferred embodiments, the scaffold comprises a biodegradable polymer, such that the scaffold degrades in vivo regardless of whether the reporter molecule is cleaved from the scaffold. By providing a biodegradable scaffold, accumulation of intact active sensors remaining in the body and any associated immune response or unintended effects can be minimized.

[0087] Other biocompatible polymers include PEG, PVA, and PVP, which are all commercially available. PVP is a non-ionic hydrophilic polymer with an average molecular weight of about 10,000 to 700,000 and a chemical formula of (C6H9NO)[n]. PVP is also known as poly[l-(2-oxo-l-pyrrolidinyl)ethylene]. PVP is non-toxic, highly hygroscopic, and readily soluble in water or organic solvents.

[0088] Polyvinyl alcohol (PVA) is a polymer made from polyvinyl acetate by replacing the acetate groups with hydroxyl groups and has a chemical formula of (CH2CHOH)[n]. Most polyvinyl alcohols are soluble in water.

[0089] Polyethylene glycol (PEG), also known as poly(oxyethylene) glycol, is a condensation polymer of ethylene oxide and water. PEG refers to a compound that includes repeating ethylene glycol units. The structure of PEG can be represented as H-(0-CH2-CH2)n-OH. PEG is a hydrophilic compound that is biologically inert (i.e., non-immunogenic) and generally considered safe for administration to humans.

[0090] When PEG is attached to a particle, it provides advantageous properties such as improved solubility, extended circulation lifetime, stability, protection from proteolytic degradation, reduced cellular uptake by macrophages, and lack of immunogenicity and antigenicity. PEG also has high flexibility and provides bioconjugation and surface treatment to particles in the absence of steric hindrance. PEG can be used for chemical modification of biologically active compounds such as peptides, proteins, antibody fragments, aptamers, enzymes, and small molecules to adjust the molecular properties of the compounds according to specific applications. Additionally, PEG molecules can be functionalized by chemically adding various functional groups to the ends of the PEG molecule, for example, amine-reactive PEG (BS(PEG)n) or thiol-reactive PEG (BM(PEG)n).

[0091] The scaffold can be a biocompatible scaffold, such as a scaffold comprising polyethylene glycol (PEG). The biocompatible scaffold can comprise a plurality of subunits of covalently linked polyethylene glycol maleimide (PEG-MAL), such as an 8-arm PEG-MAL scaffold. The PEG-containing scaffold can be selected because it is biocompatible, inexpensive, readily commercially available, taken up by the reticuloendothelial system (RES) in very small amounts, and exhibits a number of favorable behaviors. For example, PEG scaffolds can inhibit cellular uptake of particles by a variety of cell types, such as macrophages, which can help to properly distribute to specific tissues and increase residence time in the tissue.

[0092] 8-arm PEG-MAL is a multi-arm PEG derivative with a maleimide group at the end of each of its eight arms, which are attached to a hexaglycerol core. The maleimide groups selectively react with free thiol, SH, mercapto, or hydrosulfuryl groups to form stable carbon-sulfur bonds via Michael addition reactions. Each arm of the 8-arm PEG-MAL scaffold can be conjugated to a peptide, for example, via maleimide-thiol coupling or an amide bond.

[0093] The PEG-MAL scaffold can have a variety of sizes, such as a 10 kDa scaffold, a 20 kDa scaffold, a 40 kDa scaffold, or a scaffold larger than 40 kDa. The hydrodynamic diameter of a PEG scaffold in phosphate buffered saline (PBS) can be determined by various methods known in the art, for example, by dynamic light scattering. Using such techniques, the hydrodynamic diameter of a 40 kDa PEG-MAL scaffold was measured to be approximately 8 nm. In a preferred embodiment, a 40 kDa PEG-MAL scaffold is provided as the scaffold when the protease activity sensor or dual comparator is administered subcutaneously, as the scaffold readily diffuses into the systemic circulation but is not readily cleared by the reticuloendothelial system.

[0094] The size of the PEG-MAL scaffold affects the distribution and residence time of the protease activity sensor or dual comparator in vivo, as particles with a diameter less than about 5 nm can be efficiently cleared by the kidneys in vivo, even without proteolytic cleavage. In addition, particles with a diameter greater than about 10 nm are typically drained into the lymphatic vessels. In one example, when a 40 kDa 8-arm PEG-MAL scaffold is administered intravenously, the scaffold does not clear through the kidneys into the urine.

[0095] Protease activity sensors or biological comparators can include cyclic peptides that are structurally resistant to non-specific proteolysis and degradation in the body. The cyclic peptides can include protease-specific substrates or pH-sensitive bonds that enable otherwise unreactive cyclic peptides to release reactive reporter molecules in response to the presence of enzymes discussed herein. Cyclic peptides can need to be cleaved at multiple cleavage sites to increase specificity. The multiple sites can be specific for different proteases. Polycyclic peptides containing 2, 3, 4, or more cyclic peptide structures can be used with various combinations of linearizing or releasing functional peptides or other molecules required for enzymatic or environmental conditions. Cyclic peptides can include ester peptides in which hydrolysis of one or more ester bonds releases a linearized peptide. Such peptides can be used to modulate the timing of peptide release in an environment such as plasma.

[0096] FIG. 8 An exemplary protease activity sensor is shown that includes cyclic peptides 801 with protease-specific substrates 809 and a stable cyclization linker 803. The protease-specific substrates 809 are each cleaved by a different protease and can include 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. X6 can be isoleucine. These amino acids can vary between substrates in order to be specific for different proteases. The N-terminus and C-terminus coupled to the cyclization linker 803 include cyclization residues 805. The peptides can be designed to address considerations such as protease stability, steric hindrance around the cleavage site, rigidity / flexibility of the macrocycle structure and peptide chain, etc. The type and number of spacer residues 807 can be selected to address and vary many of these properties by changing the spacing between various functionalization sites of the cyclic peptide. The positioning and selection of the cyclization linker and cyclization residues also affect the above considerations. Adjustment domains (such as PEG), reporter molecules (such as FAM), and quenchers can be included in the cyclic peptides.

[0097] Adjustment domains can include ligands to help target them to specific tissues or organs. When administered to a subject, protease activity sensors or biological comparators are transported in the body by various routes depending on how they enter the body.

[0098] Cell surface receptors are membrane anchored proteins that bind to ligands on the extracellular surface. In one example, the ligand can bind to a ligand-gated ion channel, which is an ion channel that opens in response to the binding of a ligand. The ligand-gated ion channel spans the cell membrane with a hydrophilic channel in the middle. In response to a ligand that binds to the extracellular domain of the channel, the structure of the protein changes so that certain particles or ions can pass through. By providing an adjustment domain that includes a ligand that is present on the surface of a cell, the protease activity sensor or biological comparator has a greater chance of reaching and entering a particular cell to detect enzyme activity within these cells.

[0099] By providing an adjustment domain, the distribution of the protease activity sensor or biological comparator can be altered, as the ligand can target specific cells or specific tissues in the subject through the binding of the ligand to the cell surface protein on the target cell. The ligand of the adjustment domain can be selected from the group consisting of small molecules, peptides, antibodies, antibody fragments, nucleic acids, and aptamers. The ligand can also facilitate the accumulation of the protease activity sensor or biological comparator in a particular tissue type.

[0100] When the protease activity sensor or biological comparator is administered to a subject, they can be recognized by the immune system as a foreign substance and suffer immune clearance, never reaching the specific cells or specific tissues where the specific enzyme activity can release the reporter molecule. Furthermore, the generation of an immune response can destroy the purpose of monitoring the immune response sensitive activity. To inhibit immune detection, it is preferred to use a biocompatible scaffold to avoid inducing an immune response, for example, the biocompatible scaffold can include one or more subunits of polyethylene glycol maleimide. Furthermore, the molecular weight of the polyethylene glycol maleimide scaffold can also be altered to facilitate transport in the body and prevent clearance by the reticuloendothelial system. Through such alterations, the distribution and residence time in the body or in a particular tissue can be improved.

[0101] In various embodiments, the protease activity sensor or biological comparator can be designed to facilitate diffusion across the cell membrane. A hydrophobic chain can also be provided as an adjustment domain to facilitate diffusion across the cell membrane.

[0102] The adjustment domain can include any suitable hydrophobic chain that facilitates diffusion, for example, fatty acid chains (including neutral, saturated, (poly / mono)unsaturated fats and oils (monoglycerides, diglycerides, triglycerides)), phospholipids, sterols (steroids), animal sterols (cholesterol), waxes, and fat-soluble vitamins (vitamins A, D, E, and K).

[0103] The adjustment domain can comprise a cell penetrating peptide. Cell penetrating peptides (CPPs) are short peptides that facilitate cellular uptake. CPPs preferably have an amino acid composition that includes a relatively high abundance of positively charged amino acids (such as lysine or arginine) or have a sequence that includes 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 Discov Today 17:850-860, incorporated by reference. Suitable CPPs include those known in the literature, such as Tat, R6, R8, R9, Penetratin, pVEc, RRL helix, Shuffle, and Penetramax. See 2016, Cell-penetrating peptides as tools to enhance non-injectable delivery of biopharmaceuticals, Tissue Barriers 4(2):el178369, incorporated by reference.

[0104] The protease activity sensor or biological comparator can include a biocompatible polymer as the adjustment domain to protect the activity sensor from immune detection or inhibit cellular uptake of the activity sensor by macrophages.

[0105] When a foreign substance is recognized as an antigen, the immune system can trigger an antibody response. Typically, antibodies will then attach to the foreign substance, forming an antigen-antibody complex, which is then taken up by macrophages and other phagocytic cells to clear the foreign substance from the body. Likewise, when protease activity sensors or biological comparators enter the human body, they can be recognized as antigens and subject to immune clearance, preventing them from reaching a particular tissue to detect biomarker activity. To inhibit immune detection, for example, a PEG adjustment domain can be attached to the activity sensor. PEG acts as a barrier, inhibiting the immune system from recognizing it as a foreign substance. By inhibiting immune detection, the adjustment domain can improve the residence time in the body or in a particular tissue.

[0106] Enzymes have high specificity for particular substrates through binding to a pocket that has complementarity in shape, charge, and hydrophilic / hydrophobic character to the substrate. As a result, enzymes can discriminate between very similar substrate molecules, thereby having chemical selectivity (i.e., preferring the outcome of one chemical reaction over an alternative reaction), regioselectivity (i.e., preferring one direction of chemical bond formation or cleavage over all other possible directions), and stereospecificity (i.e., reacting only to one of the stereoisomers or a subset of stereoisomers).

[0107] Steric effects are non-bonding interactions that affect the shape (i.e., conformation) and reactivity of ions and molecules, which result in steric hindrance. Steric hindrance refers to the slowing of a chemical reaction due to steric bulk, thereby affecting intermolecular reactions. Various groups of a molecule can be altered to control steric hindrance between groups, for example, to control selectivity, such as for inhibiting undesired side reactions. By providing an adjustment domain for a protease activity sensor or biological comparator, such as a spacer residue between a scaffold and a cleavage site and / or any bioconjugate residue, steric hindrance between components can be minimized to increase accessibility of the cleavage site to a particular protease.

[0108] Alternatively, steric hindrance can be used to prevent access to the cleavage site until a labile, cyclized linker (e.g., an ester linkage of a cyclic ester peptide) degrades, as described above. Such labile, cyclized linkers can be other known chemical moieties that can hydrolyze under particular conditions (e.g., pH or presence of certain analytes), which can be selected to respond to particular features of a target environment.

[0109] A protease activity sensor or biological comparator can include D-amino acids in addition to protease cleavage sites to further prevent non-specific protease activity. Other unnatural amino acids can be incorporated into the peptide, including synthetic unnatural amino acids, substituted amino acids, or one or more D-amino acids.

[0110] In some embodiments, the adjustment domain can include synthetic polymers (such as polymers of lactic and glycolic acids, polyanhydrides, polyurethanes) and natural polymers (such as alginate and other polysaccharides including dextran and cellulose, collagen, albumin and other hydrophilic proteins, zein and other prolamine and hydrophobic proteins), copolymers and mixtures thereof.

[0111] One skilled in the art will know which peptide fragments to include in a protease activity sensor or biological comparator of the present disclosure as protease substrates / cleavage sites. One can use online tools or publications to identify protease substrates / cleavage sites. For example, cleavage sites are predicted in the online database PROSPER, described in Song, 2012, PROSPER: An integrated feature-based tool for predicting protease substrate cleavage sites, PLoS One 7(11): e50300, incorporated by reference. Any composition, structure, method, or activity sensor discussed herein can include, for example, any suitable cleavage site, as well as any further arbitrary polypeptide fragments to achieve any desired molecular weight. To prevent off-target cleavage, one or any number of amino acids outside of the cleavage site can be present in any number in a mixture of D and / or L forms.

[0112] The biological sample can be any sample from a subject in which the reporter molecule can be detected. For example, the sample can be a tissue sample (such as a blood sample, a hard tissue sample, a soft tissue sample, etc.), a urine sample, a saliva sample, a mucus sample, a stool sample, a semen sample, or a cerebrospinal fluid sample.

[0113] Disclosed herein are pharmaceutical compositions comprising the disclosed protease activity sensors or dual comparators. The pharmaceutical compositions containing the protease activity sensors or dual comparators are administered by parenteral (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), transdermal (passively or using iontophoresis and electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts, and can be formulated into dosage forms suitable for each route of administration. The precise dose will vary according to a variety of factors, such as subject variables (e.g., age, immune system health, etc.), disease, and ongoing treatments.

[0114] The compositions disclosed herein, including those containing peptides and polypeptides, are administered in a form of an aqueous solution by parenteral injection. The formulations can also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided, including an effective amount of a peptide or polypeptide, and optionally including a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such compositions optionally include one or more of the following: diluents, sterile water, buffers of varying ionic content (e.g., Tris-HCl, acetate, phosphate), buffered saline of varying pH and ionic strength, and additives such as detergents and solubilizers (e.g., TWEEN 20 (poly sorbate-20), TWEEN 80 (poly sorbate-80)), antioxidants (e.g., ascorbic acid), and preservatives (e.g., thimerosal, benzyl alcohol), and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations can be lyophilized and reconstituted / resuspended prior to use. The formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0115] In some embodiments, the compositions are formulated for oral delivery. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders or granules, or incorporation of the material into a microparticulate formulation of a polymeric compound such as polylactic acid, polyglycolic acid, etc., or into a liposome. Such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance. The compositions can be prepared in liquid form, or can be prepared in dry powder (e.g., lyophilized) form. Liposomal or proteinoid encapsulation can be used to formulate the compositions. Liposomal encapsulation can be used, and the liposomes can be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). Generally, the formulation will include the peptide (or chemically modified form thereof) and inert ingredients that protect the peptide in the stomach environment and release the biologically active material in the intestine.

[0116] The pharmaceutical agents can be chemically modified so that oral delivery of the derivative is effective. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where the moiety allows uptake from the stomach or intestine into the blood stream, or direct uptake into the intestinal mucosa. It is also desirable to increase the overall stability of one or more components and to prolong the circulation time in the body. PEGylation is an exemplary 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-trioxocane.

[0117] Another embodiment provides liquid dosage forms for oral administration including pharmaceutically-acceptable emulsions, solutions, suspensions, and syrups, which can contain other components including inert diluents; auxiliary agents such as wetting agents, emulsifying agents, suspending agents, and the like; and sweetening, flavoring, and perfuming agents.

[0118] Controlled release oral formulations can be desirable. The agent can be incorporated into an inert matrix (e.g., gum) that allows release by diffusion or leaching mechanisms. Slowly eroding matrices can also be added to 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 that allows water to enter and push the drug out through a single small opening due to osmotic effects.

[0119] For oral formulations, the site of release can be the stomach, the small intestine (duodenum, jejunum, or ileum), or the large intestine. In some embodiments, release will avoid the deleterious effects of the stomach environment by protecting the agent (or derivative) or by releasing the agent (or derivative) into an environment outside the stomach, such as the small intestine. To ensure adequate resistance to gastric juices, at least a coating that is impermeable to pH 5.0 is necessary. Examples of more common inert ingredients used as enteric coatings are cellulose acetate phthalate (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 can be used as mixed films.

[0120] The disclosed immunotherapeutic agents can be applied topically. For most peptide formulations, topical administration is not as effective, but it can be effective, especially when applied to the lung, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa.

[0121] The compositions can be delivered to the lung at the same time as inhalation, and when delivered as an aerosol or in spray-dried particles with an aerodynamic diameter of less than about 5 microns, can cross the pulmonary epithelial layer into the bloodstream. A variety of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Formulations for administration to the mucosa will typically be spray-dried drug particles, which can be incorporated into tablets, gels, capsules, suspensions, or emulsions. Standard pharmaceutical excipients are available from any prescription designer.

[0122] Transdermal formulations can also be prepared. These will generally be ointments, lotions, sprays or patches, all of which can be prepared using standard techniques. Transdermal formulations can need to include a penetration enhancer.

[0123] A biological sample can be any sample from a subject in which a reporter molecule can be detected. For example, the sample can be a tissue sample (such as a blood sample, a hard tissue sample, a soft tissue sample, etc.), a urine sample, a saliva sample, a mucus sample, a stool sample, a semen sample, or a cerebral spinal fluid sample. In some aspects, the sample can be obtained from a subject’s tissue or bodily fluid or from a swab taken from the patient. The sample can include a fine needle aspirate, a biopsy, or a bodily fluid from the patient. The sample can be treated, for example, to create a suspension with an appropriate solution. Such solutions are typically balanced salt solutions, such as physiological saline, PBS, Hank’s balanced salt solution, etc., and in some cases supplemented with fetal bovine serum or other naturally occurring factors, as well as used in conjunction with acceptable low concentrations (typically 5-25 mM) of buffers. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. In preferred embodiments, the sample is a respiratory tract swab (e.g., an oral, nasal, or throat swab). The swab can be placed into a sterile tube with culture medium (e.g., Hank’s balanced salt solution). The culture medium can also include antibiotics to reduce the likelihood of bacterial contamination.

[0124] EMBODIMENT

[0125] Example 1: Granzyme B / Thrombin with-gate protease activity sensors

[0126] FIGS. 9-14 With-gate sensors are shown that sense the proteases granzyme B (GzmB) and thrombin (Thrb), which play roles in the immune system killing target cells and in blood clotting, respectively. These with-gate protease sensors are designed to release a fluorescent reporter molecule (5-FAM) only after cleavage of both protease substrates. FIG. 9 ). FIG. 9 The protease activity sensors are shown to contain a dark quencher (DABCYL), a fluorescent reporter molecule (5-FAM), substrates for GzmB and Thrb (bold, cleavage sites marked with “|”), and an azide for click conjugation to a scaffold such as a nanoparticle. Unless both proteases are present, the disulfide bond cyclizes the peptide to ensure quenching of 5-FAM. When the with conditions are met, denoted by “1,1”, 5-FAM emits fluorescence.

[0127] To validate the sensor design, the specificity of the protease substrates was tested to ensure that each substrate could only be cleaved by one selected protease. Individual protease substrates were labeled with a fluorescence quencher and dye. Cleavage assays were performed by incubating the substrates with GzmB or Thrb and monitoring the fluorescence. Indeed, the GzmB substrate was efficiently cleaved by GzmB alone ( FIG. 10 ), and the Thrb substrate was cleaved only by Thrb ( FIG. 11 ). FIGS. 10-11 Shown: Average change in fluorescence after incubating nanoparticle-conjugated GzmB substrate (450 nM) and Thrb substrate (330 nM) for 1.5 hours in the absence of proteases, 275 nM GzmB, or 33 nM Thrb, n = 3.

[0128] Next, the AND logic protease sensor was tested in cleavage assays. The reporter molecule emits a high fluorescence signal when both proteases are present, but not when one protease is present alone ( FIG. 12 ). This indicates that the detectable signal only exists after cleavage by both proteases. FIG. 12 Shown: Dynamic trace of the average change in fluorescence during incubation of nanoparticle-conjugated GzmB / Thrb AND gate sensor (1 mM) with 125 nM GzmB and / or 3.7 nM Thrb for 12 hours, n = 2.

[0129] FIGS. 13-14 The AND logic sensor is very sensitive to small changes in the concentration of both proteases, even in the nanomolar range. Thus, the presence of even small amounts of a protease can be detected if the other protease is also present. FIGS. 13-14 Shown: Average change in fluorescence after incubation of the AND gate sensor with ( FIG. 13 ) 9.2 nM Thrb and different concentrations of GzmB (0-150 nM) or ( FIG. 14 ) 300 nM GzmB and different concentrations of Thrb (0-4.6 nM) for 2 hours, n = 2. For all fluorescence readings, samples were excited at 485 nm and emission was measured at 528 nm, and samples were incubated at 37 °C.

[0130] Example 2: AND protease activity sensor in living systems

[0131] FIGS. 15-17 An example of the use of the disclosed AND protease activity sensor with living systems is shown. The sensor was evaluated in an in vitro transgenic T cell killing assay, in which cytotoxic T cells taken from OT1 mice were co-incubated with target cells EL4 and EG7-OVA ( FIG. 15OT1 T cells can recognize OVA antigen and mount an immune response, so theoretically T cells would respond to EG7-OVA cells but not to EL4 cells. Supernatants were harvested from co-incubations and the immune response was quantified by GzmB secretion using ELISA FIG. 16 GzmB secretion by OT1 cells was increased by co-incubation of OT1 cells with EG7-OVA cells but not with EL4 cells, as confirmed by their concentration measured by ELISA after overnight co-incubation with target cells at 37°C, n=3.

[0132] We then performed a cleavage assay with a NAND sensor using supernatants as GzmB source and adding Thrb. FIG. 17 Dynamic traces showing the average change in fluorescence during 12 hours of incubation of nanoparticle-conjugated GzmB / Thrb with the NAND protease activity sensor (1 mM) with 10% supernatants from EL4 and EG7-OVA (after co-incubation) and / or 3.7 nM Thrb, n=3. Data for EL4 and EG7-OVA were normalized by subtracting traces with trace amounts of supernatant from OT1 alone, and data for EL4+Thrb and EG7-OVA+Thrb were normalized by subtracting traces with trace amounts of supernatant from OT1 and Thrb alone. High signal was only obtained when adding Thrb to GzmB-high OT1 / EG7-OVA supernatants, proving that the NAND protease activity sensor can accurately detect biological activity based on Boolean logic.

[0133] Example 3: NAND protease activity sensor for monitoring efficacy and response of therapeutic means

[0134] FIGS. 18-21 It is shown that the NAND protease sensor can be used for monitoring efficacy and response of therapeutic means. Cancer immunotherapy is limited by its off-target effects, so non-invasive detection of immune activity within tumors would greatly improve treatment monitoring. Moreover, monitoring immune activity in patients alone cannot confirm the efficacy of immunotherapy, as the immune system can be activated against other pathologies (e.g. viral infections). Therefore, the NAND logic sensor can improve the specificity of treatment monitoring by sensing GzmB and tumor-associated proteases. Matrix metalloproteinases (MMPs) are a class of proteases that are highly upregulated in multiple cancers, as they play a role in extracellular matrix remodeling, which facilitates tumor growth, invasion and metastasis.

[0135] FIG. 18 It is shown that the NAND protease activity sensor probes GzmB and MMPs. MMP substrates were designed according to the literature and tested to ensure that the substrates can be cleaved by different pure MMPs FIG. 19 FIG. 19 ​The average change in fluorescence is shown for the dynamic trace during incubation of MMP substrate (5 mM) with 100 nM of different MMPs for 75 minutes, n = 2. The substrate is cleaved with high activity by MMP1, MMP8 and MMP13.

[0136] The cleavage assay was then performed with the GzmB / MMP and gate sensor, testing the probe with each MMP with and without GzmB ( FIG. 20 ). Addition of GzmB to all three MMPs resulted in a significant increase in fluorescence signal over 2-fold, confirming that the probe follows the expected Boolean logic.

[0137] Next, the protease activity sensor was incubated with GzmB and MMP8 or one of two viral proteases (West Nile virus NS3 protease and Tobacco Etch virus protease) to show that the gate sensor can distinguish between immune activity in tumors and viral infection ( FIG. 21 ). The sensor with viral proteases produced a lower signal compared to MMP8.

[0138] FIGS. 20-21 The average change in fluorescence is shown for the dynamic trace after incubation of the GzmB / MMP and gate sensor (5 mM) with (7C) 300 nM GzmB and / or 100 nM of different MMPs or (7D) 300 nM GzmB and 100 nM West Nile virus NS3 protease, Tobacco Etch virus protease or MMP8 for 1.5 hours, n = 3.

[0139] Example 4: Exemplary gated biological comparator

[0140] The concept of a gated logic sensor to a system is not limited to the implementation of a protease activity sensor. The present disclosure provides a gated biological comparator consisting of a liposome in a peptide cage, such as the one formed by the MMP substrate. The liposome encapsulates a fluorescent reporter molecule, so the peptide cage must be cleaved and the liposome must be perforated by a perforin, a cytolytic protein used by T cells, for the reporter molecule to be released. The results show that by placing the fluorescent reporter molecule in a liposome, the perforin can perforate the liposome and the fluorescent reporter molecule is released after perforation ( FIG. 22 ). The results also confirm that MMP9 can cleave the MMP substrate by a cleavage assay ( FIG. 22 ). FIG. 22 A fluorescence assay is shown for the opening of naked liposomes with perforin (top) and cleavage of a peptide substrate with protease MMP9 (bottom).

[0141] Next, the biological comparator in a peptide cage was tested with perforin, MMP9 or both proteins ( FIG. 23). Only the simultaneous presence of both proteins resulted in a high signal, indicating that both proteins are necessary to release the reporter molecule in the biological comparator. FIG. 23 A fluorescence experiment showing the increase in signal of the biological comparator opened by signal proteases (MMP9) and perforin is shown.

[0142] Finally, the results from T cell killing assays show that the biological comparator can only be opened when an immune response is present and MMP9 is present. FIG. 24 An experimental schematic is shown depicting the production of MMP9 and perforin in the context of antigen-specific killing. T cells were harvested from OT1 mice and incubated with target tumor cells (EG7) secreting MMP9 and off-target cells. FIG. 25 A T cell killing assay is shown that measures the increase in signal from a biological circuit capable of detecting T cell killing and tumor activity.

[0143] While in the foregoing specification this application has been described in relation to certain embodiments thereof, and many details have been set forth for the purpose of illustration, it will be apparent to those skilled in the art that the application is not limited to the foregoing embodiments and that many substitutions, modifications, and changes can be made by those skilled in the art without departing from the spirit and scope of the present application. It is therefore desired to be secured herein a place in the art solely based upon the prior description and examples.

[0144] All references cited herein are incorporated by reference in their entirety. The application may, however, be embodied in other specific forms without departing from the spirit or essential attributes of the application. Accordingly, reference should be made to the following claims, rather than to the foregoing specification, as indicating the scope of the application.

Claims

1. A method for determining the activity of a protease in a biological sample, comprising: A protease activity sensor comprising a first protease substrate and a second protease substrate is provided for the biological sample, wherein each of the first and second protease substrates contains a protease cleavage site. The first protease substrate is cleaved by a first protease, and the second protease substrate is cleaved by a second protease. When both the first protease substrate and the second protease substrate are cleaved, the protease activity sensor provides a detectable signal through a reporter molecule, which is released through protease cleavage. Detect the presence of the detectable signal in the sample; as well as The activities of the first and second proteases are determined based on the presence of the detectable signal in the sample. The method described therein is a non-diagnostic method, and The first protease is granzyme B, the second protease is thrombin, and the protease activity sensor includes the sequence K-DABCYL-CGIEFD∣SGG-K(5-FAM)-GG-fPR∣SGGGC-K-Azide; or the first protease is granzyme B, the second protease is a metalloproteinase, and the protease activity sensor includes the sequence DABCYL-CGIEFD∣SGG-K(5-FAM)-dG-APAA∣LRAAGGC-K-Azide; where "∣" represents a cleavage site.

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

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

4. The method of claim 3, wherein the first protease substrate and the second protease substrate are conjugated with a fluorescence quencher, and the cleavage of the first protease substrate and the second protease substrate allows 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 bound to the 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 bound to the nanoparticles.

9. The method of claim 1, wherein determining the activity of the first and second proteases in the biological sample indicates protease dysregulation.

10. A composition comprising a protease activity sensor, wherein the protease activity sensor comprises: A first protease substrate and a second protease substrate, wherein each of the first protease substrate and the second protease substrate contains a protease cleavage site. The first protease substrate is cleaved by a first protease, and the second protease substrate is cleaved by a second protease. When both the first and second protease substrates are cleaved, the protease activity sensor provides a detectable signal via a reporter molecule, which is released through protease cleavage. The first protease is granzyme B, the second protease is thrombin, and the protease activity sensor includes the sequence K-DABCYL-CGIEFD∣SGG-K(5-FAM)-GG-fPR∣SGGGC-K-Azide; or the first protease is granzyme B, the second protease is a metalloproteinase, and the protease activity sensor includes the sequence DABCYL-CGIEFD∣SGG-K(5-FAM)-dG-APAA∣LRAAGGC-K-Azide, where "∣" represents a cleavage site.

11. The composition of claim 10, further comprising a biological sample.

12. The composition of claim 10, wherein the protease activity sensor is conjugated to a reporter molecule that provides the detectable signal.

13. The composition of claim 12, wherein the reporter molecule is a fluorescent molecule.

14. The composition of claim 13, wherein the first protease substrate and the second protease substrate are conjugated with a fluorescence quencher and cleavage of the first protease substrate and the second protease substrate allows the fluorescent molecule to provide a detectable signal.

15. The composition of claim 10, wherein the protease activity sensor is a cyclic peptide.

16. The composition of claim 10, wherein the protease activity sensor is bound to the scaffold.

17. The composition of claim 16, wherein the scaffold is a nanoparticle.

18. The composition of claim 17, wherein a plurality of protease activity sensors are bound to the nanoparticles.

19. A protease activity sensor, the protease activity sensor comprising the sequence K-DABCYL-CGIEFD∣SGG-K(5-FAM)-GG-fPR∣SGGGC-K-Azide, or the sequence DABCYL-CGIEFD∣SGG-K(5-FAM)-dG-APAA∣LRAAGGC-K-Azide, wherein "∣" is a cleavage site.

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