US20190345534A1 - Methods And Compositions For Noninvasive Detection Of Organ Transplant Rejection - Google Patents
Methods And Compositions For Noninvasive Detection Of Organ Transplant Rejection Download PDFInfo
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- US20190345534A1 US20190345534A1 US16/337,886 US201716337886A US2019345534A1 US 20190345534 A1 US20190345534 A1 US 20190345534A1 US 201716337886 A US201716337886 A US 201716337886A US 2019345534 A1 US2019345534 A1 US 2019345534A1
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Definitions
- Embodiments of the present disclosure relates generally to methods and compositions for noninvasive detection of transplant rejection, immune conditions related to T cell cytotoxicity (such as for example and not limitation graft versus host disease (GvHD), autoimmune diseases, and immuno-oncology), and sensing T cell cytotoxicity (e.g., to predict treatment efficacy in patients being treated with cancer immune therapies such as checkpoint blockade inhibitors or CAR T cell therapies), and more specifically to compositions comprising scaffolds (such as for example and not limitation, protein scaffolds, polymer scaffolds, and particles (e.g., a microparticle or nanoparticle)) linked to protease-specific detectable peptides that can be administered to transplant recipients and used to detect acute and chronic transplant rejection by cleavage of the composition via the accumulation of the detectable peptides locally at the site of cleavage and/or in a bodily fluid (such as for example and not limitation, urine, lymphatic fluid, blood, plasma, and/or saliva).
- a bodily fluid such
- Organ transplantation remains the single most effective treatment for end-stage organ failure, and early detection of transplant rejection is critical for managing immunosuppression and the long-term survival of recipients (1, 2).
- ACR acute cellular rejection
- graft damage is mediated by recipient cytotoxic CD8 T cells that are activated by alloantigens displayed by antigen presenting cells (APC) and target donor cells for killing (3, 4).
- ACR episodes may appear at any time during the life of the graft even years after immunological quiescence (4), ACR can be effectively treated with anti-rejection drugs that target T cells (e.g., thymoglobulin or anti-CD3 antibodies).
- the ability to measure the level of anti-graft T cell responses at an early stage of ACR plays an indispensable role in managing graft health and function (5).
- the gold standard for diagnosing ACR is the core tissue biopsy, but this procedure is invasive, subject to sampling error (tissue specimen typically represents ⁇ 1/10,000th the volume of the organ), and associated with patient morbidity (6).
- Noninvasive approaches include measuring biomarkers that indicate organ dysfunction, such as blood urea nitrogen (BUN) and serum creatinine for kidney allografts (7, 8), or biomarkers associated with allograft cell death, such as sequencing cell-free donor-derived DNA from the blood of heart transplant patients (9). These biomarkers indicate graft health at stages that are downstream of cytotoxic T cell activity; noninvasive methods that directly measure anti-graft immune activity as early biomarkers of ACR are needed.
- BUN blood urea nitrogen
- serum creatinine for kidney allografts
- biomarkers associated with allograft cell death such as sequencing cell-free donor-derived DNA
- recipient cytotoxic T cells kill allograft cells by releasing cytolytic granules containing perforin to permeate target cell membranes and the serine protease granzyme B (GzmB) to induce apoptosis (4, 10).
- GzmB serine protease granzyme B
- Noninvasive approaches to monitor anti-graft lymphocyte activity include work that showed a correlation between RNA transcript levels of GzmB, perforin, and Fas ligand from patient urine to ACR (13, 14).
- the activity of GzmB is highly contextual and dependent on the presence of endogenous inhibitors; for example, previous work showed that higher levels of the serpin protease inhibitor, PI-9, was present in kidney allografts with mild compared to severe ACR (15).
- Chronic rejection of transplanted tissue is also an issue in successful organ transplantation.
- Methods of detecting, diagnosing, and monitoring chronic rejection in transplant recipients are also needed; preferably, such methods are noninvasive.
- Proteases that are active during chronic rejection include but are not limited to proteases involved in T cell killing (such as for example and not limitation, Granzyme A and Granzyme B), T cell activation (such as for example and not limitation, MALT1, Caspase 8, Calpain 2, and Cathepsin X) (50), apoptosis (such as for example and not limitation, Caspase 3 and Caspase 8), complement activation (such as for example and not limitation, Cls, Clr, MASP2, Factor I, and Factor D) (51), fibrosis (such as for example and not limitation, ADAMTS1, MMP2, and MMP9) (52, 53), and inflammation (such as for example and not limitation, elastase, cathepsin G, PR-3, thrombin,
- T cell cytotoxicity such as for example and not limitation graft versus host disease (GvHD), autoimmune diseases, and immuno-oncology
- sensing T cell cytotoxicity e.g., to predict treatment efficacy in patients being treated with cancer immune therapies such as checkpoint blockade inhibitors or CAR T cell therapies.
- the method of detection may take advantage of the nature of the composition, wherein the composition comprises a detectable peptide containing a protease cleavage site linked or coupled to a scaffold, wherein the detectable peptide accumulates locally and/or in a bodily fluid due to the protease being active during acute or chronic rejection.
- the detectable peptide accumulates locally and/or in a bodily fluid due to the protease being active during acute or chronic rejection.
- T cell cytotoxicity such as for example and not limitation graft versus host disease (GvHD), autoimmune diseases, and immuno-oncology
- sensing T cell cytotoxicity e.g., to predict treatment efficacy in patients being treated with cancer immune therapies such as checkpoint blockade inhibitors or CAR T cell therapies
- Embodiments of the present disclosure relate generally to such methods and compositions and more specifically to compositions that can comprise scaffolds linked to detectable protease-specific peptides that can be administered to transplant recipients and used to detect acute and chronic transplant rejection by cleavage of the composition via the accumulation of the detectable peptide locally at the site of cleavage and/or in a bodily fluid (such as for example and not limitation, urine, lymphatic fluid, blood, plasma, and/or saliva).
- a bodily fluid such as for example and not limitation, urine, lymphatic fluid, blood, plasma, and/or saliva.
- the disclosure provides an activity-based nanosensor comprising: a scaffold; a linker coupled to the scaffold; at least one peptide substrate coupled to the linker; and a detectable reporter domain coupled to the at least one peptide substrate.
- the scaffold is selected from the group consisting of a nanoparticle, a nanostructure, a microparticle, a protein, a sugar, a nucleic acid-based scaffold, an imaging contrast agent, and a polymer.
- the scaffold is configured to prevent renal clearance of the substrate.
- the scaffold is a nanoparticle having a diameter from about 3 nm to about 2 microns.
- the scaffold is an iron oxide nanoparticle.
- the linker comprises a thiol group.
- the linker comprises at least one cysteine residue.
- the at least one peptide substrate comprises a target protease cleavage sequence.
- the at least one peptide substrate comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs 2-136.
- the target protease is selected from the group consisting of T cell proteases, complement proteases, fibrosis proteases, and inflammation-related proteases.
- the target protease is selected from the group consisting of Granzyme B, Granzyme A, MALT1, Caspase 8, Calpain 2, Cathepsin X, Cls, Clr, MASP2, Factor I, Factor D, ADAMTS1, MMP2, MMP9, elastase, cathepsin G, PR-3, thrombin, kallikrein 1, kallikrein 6, tryptase, and chymase.
- the reporter domain is selected from the group consisting of a fluorophore, a luminescent reporter, a ligand encoded reporter, a mass spectrometry tag, a contrast agent for imaging, a PET-detectable domain, and a nucleic acid tag.
- the reporter domain is a fluorescent reporter.
- the linker is coupled to the scaffold domain via a first spacer.
- the at least one peptide substrate is coupled to the linker via a second spacer.
- the reporter domain is coupled to the substrate via a third spacer.
- At least one of the first, second, and third spacers comprise a GGS amino acid sequence.
- the disclosure provides a method of diagnosing acute organ rejection in a transplant recipient subject comprising:
- a nanosensor comprising: a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the substrate;
- step (a) comprises intravenously administering the nanosensor to the subject.
- the bodily fluid is selected from the group consisting of urine, blood, lymphatic fluid, plasma, and saliva.
- the bodily fluid is urine.
- the reference is selected from the group consisting of a sample taken from the subject before receiving the transplanted tissue and a sample taken from a control subject.
- the therapeutic agent is configured to treat acute organ rejection.
- FIG. 1 Granzyme B sensing synthetic biomarkers detect onset of acute allograft rejection by amplifying detection signals into urine.
- Nanoparticles coated with GzmB substrates are intravenously administered and accumulate in allograft tissues.
- GzmB secreted by alloreactive CD8 T cells cleaves the peptide substrates on nanoparticle surface, which triggers the pharmacokinetic switch and release of fluorescent reporters into urine.
- Urinary signals are quantified as early stage biomarkers of acute cellular rejection.
- FIGS. 2A-2F Synthetic biomarkers are sensitive and specific to proteolytic cleavage by GzmB.
- FIG. 2A Synthetic biomarkers consist of PEG-coated IONPs functionalized with GzmB substrates. In the presence of GzmB, peptide substrates are cleaved and fluorescent reporters are released into solution, increasing sample fluorescence.
- FIG. 2C In vitro protease activity assays showing normalized fluorescence of synthetic biomarker samples after incubation with GzmB or key proteases.
- FIG. 2D Activity assays showing normalized fluorescence of synthetic biomarker samples in mouse plasma upon addition Ca2+ to initiate coagulation cascade or addition of GzmB.
- FIG. 2E Activity assays showing normalized fluorescence of synthetic biomarker samples in control serum after addition of heat aggregated gamma globulin (HAGG) to initiate complement cascade or addition of GzmB.
- MAC membrane attack complex
- FIGS. 3A-3I Sensing GzmB during cytotoxic activity of alloreactive T cells.
- FIG. 3A After upregulating expressing, activated CD8 OT1 cells secrete GzmB to mediate apoptosis of EG7-OVA target cells.
- FIG. 3B Flow cytometry plots of GzmB activity within EG7-OVA and EL4 target cells after co-cultured with OT1 T cells.
- FIG. 3C Quantified plot of flow analysis showing percent of EG7-OVA and EL4 cells having intracellular GzmB activity.
- FIG. 3E Synthetic biomarkers sense GzmB secreted in cocultures of OT1 T cells and EG7-OVA target cells.
- FIG. 3F T cell activity assays showing fluorescence of synthetic biomarkers in co-culture supernatants of OT1 T cells with EG7-OVA or EL4 target cells.
- FIG. 3G Quantified plot of T cell activity assays showing fitted value of initial cleavage velocities.
- FIG. 3H Synthetic biomarkers sense GzmB secreted in during alloreactive T cell killing.
- FIG. 3I T cell activity assays showing normalized fluorescence of synthetic biomarkers in co-culture supernatants of T cells isolated from skin graft mice with target cells from BALB/c donor mice.
- FIGS. 4A-4G Granzyme B is upregulated at the onset of acute cellular rejection.
- FIG. 4A Schematic of skin graft mouse model of acute allograft rejection.
- FIG. 4B Pictures of skin grafts showing morphological features of rejection that begin to appear on day 9 post-transplant.
- FIG. 4D Immunohistochemistry staining of GzmB in graft and healthy skin tissues from mice bearing allo- or iso-grafts.
- FIG. 4G Flow analysis of GzmB and CD44 expression in CD8+ T cells isolated from the spleens and draining lymph nodes of mice bearing allo- or iso-grafts on days 5, 7, and 9 post-transplant.
- FIGS. 5A-5I Urine analysis discriminates acute cellular rejection with high sensitivity and specificity.
- FIG. 5A Mice bearing both allo- and iso-grafts on the same animal are given surface-labelled synthetic biomarkers for biodistribution studies.
- FIG. 5B Top, photograph of mice bearing both skin grafts. Bottom, near infrared fluorescent images showing synthetic biomarkers accumulation in skin grafts.
- FIG. 5E Timeline of urinalysis study. Mice bearing either skin allografts or isografts are administered synthetic biomarkers 4 days before and 7 days after transplant surgeries.
- FIG. 5G Whole mouse fluorescent image after administration of GSBs showing strong signal from the bladders of mice bearing allografts.
- FIG. 6 Optimization of GzmB peptide substrate.
- FIGS. 7A-7B In vitro characterization of synthetic biomarkers.
- FIG. 7A Dynamic light scattering (DLS) analysis showing size distribution of synthetic biomarkers in PBS or mouse plasma.
- FIGS. 8A-8B Upregulation of intracellular GzmB expression in transgenic T cells
- FIG. 8A Activated OT1 CD8 T cells upregulated GzmB expression after coincubation with EG7-OVA target cells but not with EL4 target cells.
- FIG. 8B Flow cytometry staining of intracellular GzmB and T cell activation marker CD44 in CD8 T cells after coculturing activated OT1 T cells with EG7-OVA target cells or EL4 control cells.
- FIG. 9 Morphology of skin allografts and isografts during the course of rejection. Photographs of skin allografts and isografts on days 7, 9, 11, and 13 post-transplant. Signs of rejection began to appear in allografts at around day 9.
- FIGS. 10A-10B Upregulation of CD8-expressing cells in skin allografts.
- FIG. 10A Immunohistochemistry staining of GzmB in graft and healthy skin tissues from mice bearing allo- or iso-grafts.
- FIG. 11 Passive accumulation of synthetic biomarkers in skin allografts.
- Top panel photograph of excised allografts, isografts, and healthy skin from mice bearing both grafts on the same animal.
- Bottom panel near infra-red fluorescent image showing biodistribution of synthetic biomarkers in these tissue samples.
- FIGS. 13A-13D Urine pharmacokinetics of free peptide and bare nanoparticles.
- FIG. 13A Fluorescent image showing clearance of free peptides in bladders of skin graft mice.
- FIG. 13C Fluorescent image showing clearance of bare nanoparticles in bladders of skin graft mice.
- FIGS. 14A-14B Depletion of CD8 T cells before and after transplant surgeries.
- FIG. 14A Flow cytometry analysis showing that CD8 depletion reduces the population of CD3+ CD8+ cells in secondary lymphoid organs right before and after skin graft surgeries.
- T cell cytotoxicity such as for example and not limitation graft versus host disease (GvHD), autoimmune diseases, and immuno-oncology
- sensing T cell cytotoxicity e.g., to predict treatment efficacy in patients being treated with cancer immune therapies such as checkpoint blockade inhibitors or CAR T cell therapies
- cancer immune therapies such as checkpoint blockade inhibitors or CAR T cell therapies
- Embodiments of the present disclosure relate generally to such methods and compositions and more specifically to compositions comprising scaffolds linked to detectable protease-specific peptides that can be administered to transplant recipients and used to detect acute and chronic transplant rejection by cleavage of the composition via the accumulation of the detectable peptides in a bodily fluid (such as for example and not limitation, urine, lymphatic fluid, blood, plasma, and/or saliva), locally at the site of cleavage, and/or downstream lymph nodes.
- a bodily fluid such as for example and not limitation, urine, lymphatic fluid, blood, plasma, and/or saliva
- Detecting the onset of transplant rejection is critical for the long-term health and survival of the organ recipient, yet the core biopsy remains the diagnostic gold standard despite its invasiveness, risk of morbidity, and limited predictive power.
- host CD8 T cells damage allograft tissue by releasing the protease granzyme B (GzmB) to trigger donor cell death.
- GzmB protease granzyme B
- the inventors engineered activity-based nanoprobes to sense a target protease activity, e.g., GzmB, inside the body by producing an amplified signal in host bodily fluid, e.g., urine, for detection.
- These synthetic biomarkers can comprise target protease peptide substrates conjugated to nanoparticles, preferentially accumulate in allografts and secondary lymphoid organs, and are activated during antigen-specific T cell killing.
- target protease peptide substrates conjugated to nanoparticles preferentially accumulate in allografts and secondary lymphoid organs, and are activated during antigen-specific T cell killing.
- systemic administration of synthetic biomarkers significantly elevate urine signals at the onset of ACR before features of rejection appear in graft tissue. This is a non-limiting example of a noninvasive approach and may allow routine monitoring of allograft immune health without the risk of a biopsy.
- the inventors sought to develop a noninvasive diagnostic assay to measure the activity of GzmB within allograft tissue as an early biomarker of ACR.
- This assay can enable detection of ACR before tissue damage begins, and thus can provide a way to detect ACR before a biopsy of the transplanted tissue would indicate ACR.
- This assay can also enable monitoring of ACR over time, as compositions comprising the activity-based nanosensor may be administered repeatedly over a desired timeframe to a transplant recipient, and the diagnostic assay repeated after each administration.
- an activity-based nanosensor that can be administered intravenously (i.v.) to the recipient and can be, for example, engineered to detect elevations in GzmB activity by shedding a reporter into host urine as a noninvasive biomarker of early ACR.
- the present protease-sensing synthetic biomarkers leverage enzyme turnover to locally amplify detection signals, but by contrast, these signals are further enriched from blood into other bodily fluids, for example, urine, by renal filtration.
- these biomarkers can enable local detection of protease activity (e.g., by imaging), and/or detection in a downstream lymph node. These mechanisms for signal amplification can allow synthetic biomarkers to be ultrasensitive for early stage disease (17-21).
- synthetic biomarkers In skin graft mouse models of ACR, synthetic biomarkers accumulate in allografts and in secondary lymphoid organs to produce significantly elevated urine signals in mice bearing allografts at the onset of ACR. These protease-sensing synthetic biomarkers are noninvasive, predictive, and interact directly with host immune responses against allografts to produce amplified detection signals in urine.
- compositions of the invention can comprise detectable peptide sequences (also referred to herein as peptide substrates and/or detectable peptide substrates) that can comprise protease recognition/cleavage sites linked/coupled to scaffolds (such as for example and not limitation, protein scaffolds, polymer scaffolds, and particles (e.g., a microparticle or nanoparticle)) (e.g., activity-based nanosensors), wherein the peptide-scaffold conjugate can be capable of detecting protease activity in vivo and allowing noninvasive detection and/or monitoring of physiological processes.
- detectable peptide sequences also referred to herein as peptide substrates and/or detectable peptide substrates
- scaffolds such as for example and not limitation, protein scaffolds, polymer scaffolds, and particles (e.g., a microparticle or nanoparticle)
- the peptide-scaffold conjugate can be capable of detecting protease activity in vivo and allowing noninvasive detection
- the detectable peptide sequences can be released at the site of cleavage, which can produce a localized signal (which can be detected at the cleavage site), and can accumulate in draining lymph nodes, blood, urine, and other bodily fluids where they can be detected (e.g., by methods as described in US20140303014 and US2014/0363833, each of which is incorporated herein by reference).
- the term “and/or” may mean “and,” it may mean “or,” it may mean “exclusive-or,” it may mean “one,” it may mean “some, but not all,” it may mean “neither,” and/or it may mean “both.”
- the term “or” is intended to mean an inclusive “or.”
- Ranges may be expressed herein as from “about” or “approximately” or “substantially” one particular value and/or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and/or to the other particular value. Further, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art.
- “about” can mean a range of up to ⁇ 20%, preferably up to ⁇ 10%, more preferably up to ⁇ 5%, and more preferably still up to ⁇ 1% of a given value.
- the term can mean within an order of magnitude, preferably within 2-fold, of a value.
- substantially free of something can include both being “at least substantially free” of something, or “at least substantially pure”, and being “completely free” of something, or “completely pure”.
- the term “subject” or “patient” refers to mammals and includes, without limitation, human and veterinary animals. In a preferred embodiment, the subject is human.
- the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
- the term “therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that when administered to a subject for treating (e.g., preventing or ameliorating) a state, disorder or condition, is sufficient to effect such treatment.
- the “therapeutically effective amount” will vary depending on the compound or bacteria or analogues administered as well as the disease and its severity and the age, weight, physical condition and responsiveness of the mammal to be treated.
- the term “combination” of a composition according to the present disclosure and at least a second pharmaceutically active ingredient means at least two, but any desired combination of compounds can be delivered simultaneously or sequentially (e.g., within a 24 hour period).
- compositions according to the disclosure are used to refer to administration of a composition according to the disclosure and another therapeutic agent simultaneously in one composition, or simultaneously in different compositions, or sequentially (preferably, within a 24 hour period).
- compositions of the disclosure refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human).
- a mammal e.g., a human
- pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
- carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
- Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
- the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
- control level encompasses predetermined standards (e.g., a published value in a reference) as well as levels determined experimentally in similarly processed samples from control subjects (e.g., BMI-, age-, and gender-matched subjects without asthma as determined by standard examination and diagnostic methods) and/or from the subject prior to undergoing transplant surgery.
- compositions and methods of the disclosure can be used to detect proteolytic activity of proteases associated with immune conditions related to T cell cytotoxicity (e.g., graft versus host disease (GvHD), autoimmune diseases), as well as with both acute and chronic transplant rejection and with T cell cytotoxicity (e.g., to predict treatment efficacy in patients being treated with cancer immune therapies such as checkpoint blockade inhibitors or CAR T cell therapies).
- T cell cytotoxicity e.g., graft versus host disease (GvHD), autoimmune diseases
- T cell cytotoxicity e.g., to predict treatment efficacy in patients being treated with cancer immune therapies such as checkpoint blockade inhibitors or CAR T cell therapies.
- proteases include but are not limited to T cell proteases (such as for example and not limitation, Granzyme B, Granzyme A, MALT1, Caspase 8, Calpain 2, and Cathepsin X), complement proteases (such as for example and not limitation, Cls, Clr, MASP2, Factor I, Factor D), fibrosis proteases (such as for example and not limitation, ADAMTS1, MMP2, MMP9), and inflammatory proteases (such as for example and not limitation, elastase, cathepsin G, PR-3, thrombin, kallikreins 1&6, tryptase, and chymase).
- T cell proteases such as for example and not limitation, Granzyme B, Granzyme A, MALT1, Caspase 8, Calpain 2, and Cathepsin X
- complement proteases such as for example and not limitation, Cls, Clr, MASP2, Factor I, Factor D
- proteases have activities that can allow differentiation between acute (mediated by, e.g., T cell cytotoxicity) from chronic (mediated by, e.g., complement proteases and fibrosis) organ transplant rejection.
- T cell killing e.g., Granzyme B, Granzyme A
- T cell activation e.g., MALT1, Caspase 8, Calpain 2, Cathepsin X
- apoptosis e.g., Caspase 3, Caspase 8
- complement activation e.g., Cls, Clr, MASP2, Factor I, Factor D
- fibrosis e.g., ADAMTS1, MMP2, MMP9
- inflammation e.g., elastase, cathepsin G, PR-3, thrombin, kallikreins 1&6, tryptase, and chymase.
- compositions of the present disclosure can be designed to contain one or more detectable peptide sequences (also referred to herein as peptide substrates and/or detectable peptide substrates) that are capable of being recognized by the proteases and are also linked or coupled to scaffolds (such as for example and not limitation, protein scaffolds, polymer scaffolds, and particles (e.g., microparticles or nanoparticles)), and the detectable peptide sequences can accumulate in a bodily fluid (such as for example and not limitation, urine, plasma, draining lymph, blood, saliva, etc.) after being cleaved from the peptide-scaffold complex by the protease of interest.
- the composition can further comprise an optional spacer region located between the one or more detectable peptide sequences and the scaffold and adjacent either side of the linker region.
- a conjugate that can comprise one or more peptide sequences operably linked to at least one scaffold, wherein the conjugate can be recognized by a protease as described herein.
- the one or more peptide sequences and/or the scaffold of the conjugate can be capable of generating a detectable signal such that the conjugate can be visualized and tracked in real time.
- the one or more detectable peptide sequences can comprise a reporter (also referred to herein as a reporter domain) and a protease cleavage/recognition site.
- the at least one detectable peptide sequence can be cleaved, such that the reporter of the detectable peptide sequence is released.
- the reporter can be detected locally at the site of cleavage (i.e., at the site of the protease activity) or in the subject's blood or plasma (e.g., by ELISA), or can travel to one or more nearby draining lymph nodes and be detected there, or can travel to the kidney and be renally-cleared and detectable in urine.
- the reporter thus functions as a “messenger” of enzyme activity.
- the composition or conjugate can remain uncleaved, indicating that the proteases are not active.
- the reporter includes for example and not limitation, a fluorophore (e.g., for fluorescent detection), a luminescent reporter (e.g., for bioluminescent assays), a ligand encoded reporter (e.g., for detection by ELISA or other antibody detection systems), a mass tag (e.g., for detection by mass spectrometry), and a nucleic acid tag (e.g., for detection by PCR), etc.
- a fluorophore e.g., for fluorescent detection
- a luminescent reporter e.g., for bioluminescent assays
- a ligand encoded reporter e.g., for detection by ELISA or other antibody detection systems
- mass tag e.g., for detection by mass spectrometry
- a nucleic acid tag e.g., for detection by PCR
- the detectable peptide sequence can be detected by a method such as, for example and not limitation, Sanger sequencing, pyrosequencing, SOLID sequencing, massively parallel sequencing, barcoded DNA sequencing, PCR, real-time PCR, quantitative PCR, microarray analysis of the isolated nucleic acid with a gene chip, restriction fragment length polymorphism analysis, allele specific ligation, comparative genomic hybridization, microarray/microchip analysis of the isolated nucleic acid, DNA/RNA in situ hybridization, RNAse protection assay, Northern blot, reverse transcriptase PCR, quantitative PCR, quantitative reverse transcriptase PCR, quantitative real-time reverse transcriptase PCR, reverse transcriptase treatment followed by direct sequencing, flow cytometry, bead-based flow-cytometry, immunohistochemistry, ELISA, RIA, Western blot, immunoaffinity chromatography, HPLC, mass spectrometry, mass spectroscopy, protein microarray/microchip analysis, PAGE analysis, is
- the scaffold can comprise a protein scaffold (e.g., albumin, IgG, IgG Fc, antibody-based, antibody fragment-based, etc.), a polymer scaffold (e.g., PEG, PLGA), a DNA scaffold (e.g., (DNA organisms), a sugar scaffold (e.g., dextran), imaging (e.g., magnetic resonance imaging) contrast agents (e.g., gadolinium, iron oxide) and a particle (e.g., a microparticle or nanoparticle, such as for example and not limitation, nanostructures including nanofibers, nanorods, nanotubes).
- a protein scaffold e.g., albumin, IgG, IgG Fc, antibody-based, antibody fragment-based, etc.
- a polymer scaffold e.g., PEG, PLGA
- a DNA scaffold e.g., (DNA organisms)
- a sugar scaffold e.g., dextran
- imaging e.g., magnetic
- the scaffold is detectable, e.g., by fluorescence, mass spectrometry, magnetic imaging, ELISA, luminescence, etc.
- the scaffold can have a size ranging from 3 nanometers to 2 micrometers, including from 10 nanometers to 1.5 micrometers, 20 nanometers to 1 micrometer, 50 nanometers to 0.1 micrometer, and 50 nanometers to 150 nanometers.
- the scaffold can provide the composition with a longer circulation half-life, such as for example and not limitation, by preventing the composition from being trafficked to the lymph and/or being cleared from circulation due to its small size.
- the circulation half-life of the compositions of the disclosure can be at least 1 hour to 52 weeks, from 2 hours to 36 hours, from 3 hours to 24 hours, and/or 5 hours to 12 hours.
- the scaffold can be monovalent or polyvalent, meaning that it can be coupled to at least one detectable peptide sequence to 5,000 detectable peptide sequences, including from 50-100 detectable peptide sequences.
- nanoparticle includes nanoparticles as well as microparticles.
- Nanoparticles are defined as particles of less than 1.0 ⁇ m in diameter.
- a preparation of nanoparticles includes particles having an average particle size of less than 1.0 ⁇ m in diameter.
- Microparticles are particles of greater than 1.0 ⁇ m in diameter but less than 1 mm.
- a preparation of microparticles includes particles having an average particle size of greater than 1.0 ⁇ m in diameter.
- the microparticles may therefore have a diameter of at least 5, at least 10, at least 25, at least 50, or at least 75 microns, including sizes in ranges of 5-10 microns, 5-15 microns, 5-20 microns, 5-30 microns, 5-40 microns, or 5-50 microns.
- a composition of particles may have heterogeneous size distributions ranging from 10 nm to mm sizes.
- the diameter is about 5 nm to about 500 nm.
- the diameter is about 100 nm to about 200 nm.
- the diameter is about 10 nm to about 100 nm.
- the particles may be composed of a variety of materials including iron, ceramic, metallic, natural polymer materials (including lipids, sugars, chitosan, hyaluronic acid, etc.), synthetic polymer materials (including poly-lactide-coglycolide, poly-glycerol sebacate, etc.), and non-polymer materials, or combinations thereof.
- the polymers may be biodegradable (such as for example and not limitation, synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxybutyrate), poly(lactide-co-glycolide) and poly(lactide-co-caprolactone), and natural polymers such as algninate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof.
- synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes,
- these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
- the foregoing materials may be used alone, as physical mixtures (blends), or as co-polymers.
- the polymers are polyesters, polyanhydrides, polystyrenes, polylactic acid, polyglycolic acid, and copolymers of lactic and glycoloic acid and blends thereof), or non-biodegradable (such as for example and not limitation, ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof).
- the particles may be composed in whole or in part of polymers or non-polymer materials.
- Non-polymer materials may be employed in the preparation of the particles.
- Exemplary materials include alumina, calcium carbonate, calcium sulfate, calcium phosphosilicate, sodium phosphate, calcium aluminate, calcium phosphate, hydroxyapatite, tricalcium phosphate, dicalcium phosphate, tricalcium phosphate, tetracalcium phosphate, amorphous calcium phosphate, octacalcium phosphate, and silicates.
- the particles may comprise a calcium salt such as calcium carbonate, a zirconium salt such as zirconium dioxide, a zinc salt such as zinc oxide, a magnesium salt such as magnesium silicate, a silicon salt such as silicon dioxide or a titanium salt such as titanium oxide or titanium dioxide.
- a calcium salt such as calcium carbonate
- a zirconium salt such as zirconium dioxide
- a zinc salt such as zinc oxide
- a magnesium salt such as magnesium silicate
- silicon salt such as silicon dioxide or a titanium salt such as titanium oxide or titanium dioxide.
- the detectable peptide sequences may be coupled to one or more linkers to aid in joining the one or more peptide sequences to the scaffold.
- the linker may be any suitable linker for joining peptide sequences to scaffolds.
- the linker comprises a small molecule linker SIA to join primary amines on nanoparticle surface to thiols on cysteine-terminated peptides.
- the linker comprises small molecule linkers such as for example and not limitation, amine to sulfhydryl linkers (e.g., BMPS, MBS, SMCC, SMPH, SPDP, SBAP), carboxyl to amine linkers (e.g., DCC, EDC, EDAC), and/or sulfhydryl-to-carbohydrate linkers (e.g., BMPH, EMCH, MPBH).
- the linker may contain a thiol group, such as for example and not limitation, in a cysteine residue (which may be in D or L form).
- Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, peptide linkers, nucleic acid molecules, polypeptides, lipids, fatty acids, peptide nucleic acids, aptamers, DNA, RNA, leucine zippers, oligonucleotides, oligopeptides, biotin, avidin, streptavidin, haptene antibody bonds or biotin avidin bonds.
- Linkers can also be derivatives of PEG or other biocompatible polymers of different sizes. The linkers can be capable of forming covalent bonds to amino groups, carboxyl groups, or sulfhydryl groups or hydroxyl groups.
- Amino-binding linkers include reactive groups such as carboxyl groups, isocyanates, isothiocyanates, esters, haloalkyls, and the like.
- Carboxyl-binding linkers are capable of forming include reactive groups such as various amines, hydroxyls and the like.
- Sulfhydryl-binding linkers include reactive groups such as sulfhydryl groups, acrylates, isothiocyanates, isocyanates and the like.
- Hydroxyl binding groups include reactive groups such as carboxyl groups, isocyanates, isothiocyanates, esters, haloalkyls, and the like.
- an end of the linker is capable of binding to a crystalline composition formed from a Group IV metal element.
- the linker may be of any suitable length and can be varied to bring the detectable peptide sequence and the scaffold closer together or farther apart as desired.
- Exemplary short linkers may be strands of RNA, DNA, short amino acid sequences, polypeptides, fatty acids, proteins, antibodies, or other small molecules.
- the detectable peptide sequences are linked/coupled to the at least one scaffold by a thiol group, such as that of a cysteine residue (e.g., a N-terminal and/or C-terminal and/or internal cysteine residue).
- the detectable peptide sequences are linked/coupled to the scaffold by a lysine linker or residue (e.g., a N-terminal and/or C-terminal and/or internal lysine linker or residue).
- the one or more detectable peptide sequences are operably linked to the at least one scaffold through a linker or chemical linkage comprising at least one bond selected from the group consisting of: a covalent bond, an electrostatic bond and a chelation bond.
- the bond is a covalent bond, which can be a bond through a functional group selected from the group consisting of: a hydroxyl, a carboxyl, a carbonyl, a sulfhydryl, an amine, an amide, a nitrile, a nitrogen with a free lone pair of electrons, an amino acid, a thiol, a polyethylene glycol, a sulfonic acid, a sulfonyl halide, and an acyl halide.
- the bond is an amide bond formed through reaction of a carboxyl group of the at least one scaffold and an amine group of the one or more detectable peptide sequences.
- the bond is a thioether bond formed through a reaction involving the thiol group of a natural or engineered cysteine residue of the one or more detectable peptide sequences.
- the one or more peptide sequences are operably linked to at least one scaffold through a linker or chemical linkage comprising at least one chelation bond.
- the chelation bond can be formed between a metal of the scaffold and a metal-chelating ligand attached to or otherwise associated with the one or more peptide sequences.
- the metal-chelating ligand can comprise one or more naturally occurring or engineered histidine residues of the one or more detectable peptide sequences.
- the metal-chelating ligand comprises a histidine tag fused to the N-terminus or the C-terminus of the one or more peptide sequences.
- the one or more detectable peptide sequences are linked to the at least one scaffold by enzymatic linkages, such as for example and not limitation, by a bond formed by a ligase, sortase mediated linkages, HaloTag linkages, SNAP-tag linkages, CLIP-tag linkages, full-length or split intein-mediated linkages, BirA-mediated linkages, Sfp-mediated linkages, and other bioconjugation methods.
- the linker is a protein of 10-100 amino acids in length.
- the linker may be 8 nm-100 nm, 6 nm-100 nm, 8 nm-80 nm, 10 nm-100 nm, 13 nm-100 nm, 15 nm-50 nm, or 10 nm-50 nm in length.
- compositions of the invention may also comprise at least one optional spacer region located between the one or more detectable peptide sequences and the scaffold.
- the optional spacer region(s) may be located on either side of the linker, e.g., between the linker and the scaffold and/or between the linker and the one or more detectable peptide sequences.
- the spacer region can comprise a GGS amino acid sequence.
- the spacer can be a small peptide, such as for example and not limitation, GGS, GGGS, (GGGS) 2 , (GGGS) 3 , (Gly) 6 , (Gly) 8 , (EAAK) 3 , PAPAP, A(EAAAK) 3 .
- the linker can comprise primarily D-form amino acids to resist proteolytic cleavage. Spacers can also be derivatives of PEG or other biocompatible polymers of different sizes.
- the peptide sequences of the disclosure can include those that can be recognized by proteases associated with immune conditions (e.g., graft versus host disease (GvHD), autoimmune diseases), as well as with both acute and chronic transplant rejection and with T cell cytotoxicity (e.g., to predict treatment efficacy in patients being treated with cancer immune therapies such as checkpoint blockade inhibitors or CAR T cell therapies).
- the peptide sequences are recognized by Granzyme B (GzmB), such as a recombinant GzmB (SEQ ID NO: 1).
- the peptide sequences are recognized by T cell-associated proteases (such as for example and not limitation, Granzyme B, Granzyme A, MALT1, Caspase 8, Calpain 2, and Cathepsin X).
- the peptide sequences are recognized by complement-associated proteases (such as for example and not limitation, Cls, Clr, MASP2, Factor I, Factor D).
- the peptide sequences are recognized by fibrosis-associated proteases (such as for example and not limitation, ADAMTS1, MMP2, MMP9).
- the peptide sequences are recognized by inflammation-associated proteases (such as for example and not limitation, elastase, cathepsin G, PR-3, thrombin, kallikreins 1&6, tryptase, and chymase).
- the peptide sequences may contain amino acids in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the one or more detectable peptide sequences linked to the scaffold comprises a reporter, a GzmB recognition/cleavage sequence comprising any of SEQ ID NOs 2 to 81, and combinations thereof, and another peptide, protein, nucleic acid, lipid, fatty acid, oligonucleotide, oligopeptide, oligolipid, antibody, antibody fragment, aptamer and/or binding protein (including binding protein fragments), which can aid in coupling the detectable peptide to the scaffold.
- the peptide sequences comprising any of SEQ ID NOs 2 to 81 can contain amino acids in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the one or more peptide sequences comprises a GzmB recognition/cleavage sequence comprising any of SEQ ID NOs 2 to 81 and combinations thereof, and polyethylene glycol (e.g., one or more polyethylene glycol molecules).
- the polyethylene glycol may be of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000.
- the one or more detectable peptide sequences linked to the scaffold comprises a reporter, a protease recognition/cleavage sequence comprising any of SEQ ID NOs 82 to 136, and combinations thereof, and another peptide, protein, nucleic acid, lipid, fatty acid, oligonucleotide, oligopeptide, oligolipid, antibody, antibody fragment, aptamer and/or binding protein (including binding protein fragments), which can aid in coupling the detectable peptide to the scaffold.
- the peptide sequences comprising any of SEQ ID NOs 82 to 136 can contain amino acids in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the one or more peptide sequences comprises a reporter, a protease recognition/cleavage sequence comprising any of SEQ ID NOs 82 to 136 and combinations thereof, and polyethylene glycol (e.g., one or more polyethylene glycol molecules).
- the polyethylene glycol may be of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000.
- the one or more detectable peptide sequences linked to the scaffold may comprise a recognition/cleavage site for GzmB.
- the peptide may comprise an amino acid sequence comprising at least one of AIEPDGSC (SEQ ID NO: 2), ASGIEPDSGGSC (SEQ ID NO: 3), AKSKIEFDFGVKKC (SEQ ID NO: 4), AIEPDSGC (SEQ ID NO: 5), AIEPDGSSKC (SEQ ID NO: 6), AIEPDSGSKC (SEQ ID NO: 7), AKSIEPDGSSKC (SEQ ID NO: 8), AKSIEPDSGSKC (SEQ ID NO: 9), AIEFDGSC (SEQ ID NO: 10), AIEFDSGC (SEQ ID NO: 11), AIEFDSGSKC (SEQ ID NO: 12), AKSIEFDSGSKC (SEQ ID NO: 13), AIEFDSGVSKC (SEQ ID NO: 14), and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the peptide, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding each of SEQ ID NOs 2-14, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 2-14 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- polyethylene glycol of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000
- Glufib an internal lysine residue for linking
- cysteine residue at the N-terminus and/or C-terminus.
- the one or more detectable peptide sequences linked to the scaffold may comprise an amino acid sequence comprising one or more D-form amino acids, such as for example and not limitation, at least one of AIEPDGSc (SEQ ID NO: 15), AsGIEPDSGGsc (SEQ ID NO: 16), AksKIEFDFGVKkc (SEQ ID NO: 17), AIEPDSGc (SEQ ID NO: 18), AIEPDGSskc (SEQ ID NO: 19), AIEPDSGskc (SEQ ID NO: 20), AksIEPDGSskc (SEQ ID NO: 21), AksIEPDSGskc (SEQ ID NO: 22), AIEFDGSc (SEQ ID NO: 23), AIEFDSGc (SEQ ID NO: 24), AIEFDSGskc (SEQ ID NO: 25), AksIEFDSGskc (SEQ ID NO: 26), AIEFDSGVskc (SEQ ID NO: 27), and combinations thereof, including combinations with any peptide disclosed herein
- Suitable linkers may be added to the N-terminus and/or C-terminus of the peptide, and/or to residues within the one or more peptide sequences.
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding each of SEQ ID NOs 15-27, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 15-27 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- polyethylene glycol of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000
- Glufib an internal lysine residue for linking
- cysteine residue at the N-terminus and/or C-terminus.
- the one or more detectable peptide sequences linked to the scaffold comprises a GzmB recognition/cleavage sequence and a Glu-1-Fibrinopeptide B peptide EGVNDNEEGFFSAR (Glufib peptide; SEQ ID NO: 28).
- the Glufib peptide comprises one or more D-form amino acids, such as for example and not limitation, eGvndneeGffsar (lowercase letters denote D-form amino acids; SEQ ID NO: 29).
- the peptide comprises an amino acid sequence comprising at least one of AIEPDGSC (SEQ ID NO: 30), ASGIEPDSGGSC (SEQ ID NO: 31), AKSKIEFDFGVKKC (SEQ ID NO: 32), AIEPDSGC (SEQ ID NO: 33), AIEPDGSSKC (SEQ ID NO: 34), AIEPDSGSKC (SEQ ID NO: 35), AKSIEPDGSSKC (SEQ ID NO: 36), AKSIEPDSGSKC (SEQ ID NO: 37), AIEFDGSC (SEQ ID NO: 38), AIEFDSGC (SEQ ID NO: 39), AIEFDSGSKC (SEQ ID NO: 40), AKSIEFDSGSKC (SEQ ID NO: 41), AIEFDSGVSKC (SEQ ID NO: 42), aIEPDGSc (SEQ ID NO: 43), asGIEPDSGGsc (SEQ ID NO: 44), aksKIEFDFGVKkc (SEQ ID NO: 45), aIEPDSGc (SEQ ID NO:
- the peptide comprises an amino acid sequence comprising one or more of EGVNDNEEGFFSARKAIEPDGSC (SEQ ID NO: 56), EGVNDNEEGFFSARKASGIEPDSGGSC (SEQ ID NO: 57), EGVNDNEEGFFSARKAKSKIEFDFGVKKC (SEQ ID NO: 58), EGVNDNEEGFFSARKAIEPDSGC (SEQ ID NO: 59), EGVNDNEEGFFSARKAIEPDGSSKC (SEQ ID NO: 60), EGVNDNEEGFFSARKAIEPDSGSKC (SEQ ID NO: 61), EGVNDNEEGFFSARKAKSIEPDGSSKC (SEQ ID NO: 62), EGVNDNEEGFFSARKAKSIEPDSGSKC (SEQ ID NO: 63), EGVNDNEEGFFSARKAIEFDGSC (SEQ ID NO: 64), EGVNDNEEGFFSARKAIEFDSGC (SEQ ID NO: 65), EGVNDNEEGFFSARKAIEFDSGSKC (SEQ ID NO:
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding each of SEQ ID NOs 28-81, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 30-81 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- polyethylene glycol of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000
- Glufib an internal lysine residue for linking
- cysteine residue at the N-terminus and/or C-terminus.
- the one or more detectable peptide sequences linked to the scaffold comprises a recognition/cleavage site that can be recognized by another T cell-related protease, e.g., a protease involved in T cell killing and/or a protease involved in T cell activation.
- a protease involved in T cell killing include Granzyme B and Granzyme A.
- proteases involved in T cell activation include MALT1, Caspase 8, Calpain 2, and Cathepsin X.
- the one or more detectable peptide sequences linked to the scaffold comprises a Granzyme A recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising at least one of AAPVRSL (SEQ ID NO: 82), ALDPRSF (SEQ ID NO: 83), ATQNKAS (SEQ ID NO: 84) and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding each of SEQ ID NOs 82-84, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 82-84 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- polyethylene glycol of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000
- Glufib an internal lysine residue for linking
- cysteine residue at the N-terminus and/or C-terminus.
- the one or more detectable peptide sequences linked to the scaffold comprises a MALT1 recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising ACYLD (SEQ ID NO: 85) and combinations including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID NO: 85, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NO: 85 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- the disclosure further contemplates methods of making said vectors and host cells according to cloning methods well known in the art.
- the one or more detectable peptide sequences linked to the scaffold comprises a Caspase 8 recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising at least one of AIETDGS (SEQ ID NO: 86) and ALEVDCY (SEQ ID NO: 87) and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding each of SEQ ID NOs 86-87, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 86-87 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- the disclosure further contemplates methods of making said vectors and host cells according to cloning methods well known in the art.
- the one or more detectable peptide sequences linked to the scaffold comprises a Caspase 3 recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising at least one of ADEVDNK (SEQ ID NO: 116), ADEVDGV (SEQ ID NO: 117), ADEVDRD (SEQ ID NO: 118), ADEVDGV (SEQ ID NO: 119) and ALEVDCY (SEQ ID NO: 120) and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding each of SEQ ID NOs 86-87, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 86-87 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- polyethylene glycol of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000
- Glufib an internal lysine residue for linking
- cysteine residue at the N-terminus and/or C-terminus.
- the one or more detectable peptide sequences linked to the scaffold comprises a Calpain2 recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising AEPLFAERK (SEQ ID NO: 88) and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID NO: 88, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NO: 88 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- the disclosure further contemplates methods of making said vectors and host cells according to cloning methods well known in the art.
- the one or more detectable peptide sequences linked to the scaffold comprises a Cathepsin X recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising AMNPKFA (SEQ ID NO: 89) and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID NO: 89, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NO: 89 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- the disclosure further contemplates methods of making said vectors and host cells according to cloning methods well known in the art.
- the one or more detectable peptide sequences linked to the scaffold comprises a Cls, Clr, MASP2, Factor I, and/or Factor D recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising at least one of ALQRIYKC (SEQ ID NO: 90), AKSVARTLLVKC (SEQ ID NO: 91), AEEKQRIIGC (SEQ ID NO: 92), AQRQRIIGGC (SEQ ID NO: 93), ALGRGGSC (SEQ ID NO: 94), AKYLGRSYKVC (SEQ ID NO: 95), ARALERGLQDC (SEQ ID NO: 96), ASLGRKIQIC (SEQ ID NO: 97), AGLQRALEIC (SEQ ID NO: 98), AKVFMGRVYDPC (SEQ ID NO: 99), ASSTGRNGFKC (SEQ ID NO: 100), AKTTGGRIYGGC (SEQ ID NO: 101), ADPRGGSC (SEQ ID NO:
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding each of SEQ ID NOs 90-109, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 90-109 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- polyethylene glycol of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000
- Glufib an internal lysine residue for linking
- cysteine residue at the N-terminus and/or C-terminus.
- the one or more detectable peptide sequences linked to the scaffold comprises an ADAMTS1 recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising at least one of AIPENFF (SEQ ID NO: 110), AKEEEGL (SEQ ID NO: 111), ANLVYMV (SEQ ID NO: 112), and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding each of SEQ ID NOs 110-112, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 110-112 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- polyethylene glycol of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000
- Glufib an internal lysine residue for linking
- cysteine residue at the N-terminus and/or C-terminus.
- the one or more detectable peptide sequences linked to the scaffold comprises a MMP2 recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising at least one of ARLAAIT (SEQ ID NO: 113), ASLSRLT (SEQ ID NO: 114), and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding each of SEQ ID NOs 113-114, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 113-114 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- the disclosure further contemplates methods of making said vectors and host cells according to cloning methods well known in the art.
- the one or more detectable peptide sequences linked to the scaffold comprises a MMP9 recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising APLGVRGK (SEQ ID NO: 115), and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID NO: 115, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NO: 115 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- the disclosure further contemplates methods of making said vectors and host cells according to cloning methods well known in the art.
- the one or more detectable peptide sequences linked to the scaffold comprises an elastane, cathepsin G, and/or PR-3 recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising at least one of AAAPVc (SEQ ID NO: 121), AAAPAc (SEQ ID NO: 122), AAAPLc (SEQ ID NO: 123), AAAPMc (SEQ ID NO: 124), AAAPFc (SEQ ID NO: 125), and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID NOs 121-125, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 121-125 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- polyethylene glycol of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000
- Glufib an internal lysine residue for linking
- cysteine residue at the N-terminus and/or C-terminus.
- the one or more detectable peptide sequences linked to the scaffold comprises a thrombin recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising at least one of GGFPRSGGGc (SEQ ID NO: 126), AGFPRSGGGc, (SEQ ID NO: 127), and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID NOs 126-127, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 126-127 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- polyethylene glycol of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000
- Glufib an internal lysine residue for linking
- cysteine residue at the N-terminus and/or C-terminus.
- the one or more detectable peptide sequences linked to the scaffold comprises a kallikrein 1 recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising AIKFFSAc (SEQ ID NO: 128), and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID NO: 128, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NO: 128 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- the disclosure further contemplates methods of making said vectors and host cells according to cloning methods well known in the art.
- the one or more detectable peptide sequences linked to the scaffold comprises a kallikrein 6 recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising at least one of ALRQRESc (SEQ ID NO: 129), AAEFRHDc (SEQ ID NO: 130), and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID NOs 129-130, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 129-130 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- the disclosure further contemplates methods of making said vectors and host cells according to cloning methods well known in the art.
- the one or more detectable peptide sequences linked to the scaffold comprises a chymase recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising at least one of AAAPFc (SEQ ID NO: 131), AQFVLTEc (SEQ ID NO: 132), ARETYGEc (SEQ ID NO: 133), AATVYVDc (SEQ ID NO: 134), and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID NOs 131-134, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 131-134 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- polyethylene glycol of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000
- Glufib an internal lysine residue for linking
- cysteine residue at the N-terminus and/or C-terminus.
- the one or more detectable peptide sequences linked to the scaffold comprises a tryptase recognition/cleavage site, such as for example and not limitation, an amino acid sequence comprising at least one of APLDKKRc (SEQ ID NO: 135), ADKVKAQc (SEQ ID NO: 136), and combinations thereof, including combinations with any peptide disclosed herein.
- Suitable linkers may be added to the N-terminus and/or C-terminus of the one or more peptide sequences, and/or to residues within the one or more peptide sequences.
- the amino acids can be present in all L form, all D form, or a mix of L and D forms (ranging from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
- the disclosure also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID NOs 135-136, and modifications thereof (e.g., linkers) as well as vectors comprising such nucleic acids, and host cells comprising such vectors.
- the peptides comprising any of the amino acid sequences comprising SEQ ID NOs 135-136 may further comprise one or more of polyethylene glycol (of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue for linking, and/or a cysteine residue at the N-terminus and/or C-terminus.
- polyethylene glycol of any suitable molecular weight, such as for example and not limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000
- Glufib an internal lysine residue for linking
- cysteine residue at the N-terminus and/or C-terminus.
- the disclosure in some aspects involves administering to a subject, such as for example and not limitation a transplant recipient, a composition comprising at least one detectable peptide sequence coupled/linked to a scaffold as described herein, identifying a biological sample from the subject in which to detect the detectable peptide sequence and optionally collecting the sample; and, subjecting the biological sample to an analysis method in order to detect the presence of the peptide sequence.
- a detectable marker in the biological sample is indicative of an active enzyme or a substrate within the subject, and allows further diagnosis of a condition, detection of a condition, prediction of a condition, classification of the patient, and/or selection of the patient, discussed in further detail hereinbelow.
- the patient is treated with an appropriate therapeutic composition and/or method based on the results of the analysis method.
- a biological sample such as for example and not limitation, a bodily fluid containing T cells, urine, blood, lymphatic fluid, plasma, saliva, etc.
- a composition comprising at least one detectable peptide sequence linked/coupled to a scaffold as described herein.
- the sample is subjected to similar analyses as discussed herein in order to detect the presence of the peptide sequence.
- the presence of the detectable marker in the biological sample is indicative of an active enzyme or a substrate within the subject, and allows further diagnosis of a condition, detection of a condition, prediction of a condition, classification of the patient, and/or selection of the patient, discussed in further detail hereinbelow.
- the present disclosure also provides methods of diagnosing acute rejection in a patient who is a transplant recipient by using the compositions described herein, as well as methods of detecting acute rejection in such a patient, predicting acute rejection in such a patient, classifying a transplant recipient as having acute rejection of the transplanted tissue, monitoring acute rejection in a transplant recipient, selecting a transplant recipient for a clinical trial for acute rejection-related therapeutic compositions and/or methods, and methods of treating acute rejection in a transplant recipient.
- the detectable peptide sequences are released at the site of protease cleavage, producing a localized detectable signal (that can be detected at the site of cleavage or downstream of the site of cleavage), and can accumulate in draining lymph nodes, blood, urine, and other bodily fluids where they can be detected (e.g., by methods as described in US2014/0303014 and US2014/0363833, each of which is incorporated herein by reference).
- the disclosure provides a method of diagnosing acute organ rejection in a transplant recipient subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) acutely rejecting the transplant when the activity of the target protease in the sample is greater than the reference activity of the target proteas
- the disclosure provides a method of detecting acute organ rejection in a transplant recipient subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) acutely rejecting the transplant when the activity of the target protease in the sample is greater than the reference activity of the target proteas
- the disclosure provides a method of predicting acute organ rejection in a transplant recipient subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) at risk of or likely to acutely reject the transplant when the activity of the target protease in the sample is greater than the reference activity of
- the disclosure provides a method of classifying a transplant recipient subject as having or likely to have acute organ rejection comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of or likely to acutely reject the transplant when the activity of the target protease in the sample
- the disclosure provides a method of monitoring acute organ rejection in a transplant recipient subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of or likely to acutely reject the transplant when the activity of the target protease in the sample is greater than the reference activity
- steps (a)-(g) can be repeated over a period of hours to weeks after the subject has received the transplanted tissue, for example, 3 hours to three weeks after receiving the transplanted tissue. In other embodiments, steps (a)-(g) can be repeated over a period of hours to weeks after the subject has clinical signs of graft functional decline, for example, 3 hours to three weeks after the first sign of graft functional decline.
- the disclosure provides a method of selecting a transplant recipient subject for a clinical trial for an acute organ rejection therapeutic and/or prophylactic compositions and/or methods comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) suitable for the trial when the activity of the target proteas
- the disclosure provides a method of treating a transplant recipient subject for acute organ rejection comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of or likely to acutely reject the transplant when the activity of the target protease in the sample is greater than the reference activity
- the disclosure provides methods of diagnosing chronic rejection in a patient who is a transplant recipient by using the compositions described herein, as well as methods of detecting chronic rejection in such a patient, predicting chronic rejection in such a patient, classifying a transplant recipient as having chronic rejection of the transplanted tissue, monitoring chronic rejection in a transplant recipient, selecting a transplant recipient for a clinical trial for chronic rejection-related therapeutic compositions and/or methods, and methods of treating chronic rejection in a transplant recipient.
- the detectable peptide sequences are released at the site of protease cleavage, producing a localized detectable signal (that can be detected at the site of cleavage or downstream of the site of cleavage), and can accumulate in draining lymph nodes, blood, urine, and other bodily fluids where they can be detected (e.g., by methods as described in US2014/0303014 and US2014/0363833, each of which is incorporated herein by reference).
- the disclosure provides a method of diagnosing chronic organ rejection in a transplant recipient subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) chronically rejecting the transplant when the activity of the target protease in the sample is greater than the reference activity of the target proteas
- the disclosure provides a method of detecting chronic organ rejection in a transplant recipient subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) chronically rejecting the transplant when the activity of the target protease in the sample is greater than the reference activity of the target proteas
- the disclosure provides a method of predicting chronic organ rejection in a transplant recipient subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) at risk of or likely to chronically reject the transplant when the activity of the target protease in the sample is greater than the reference activity of
- the disclosure provides a method of classifying a transplant recipient subject as having or likely to have chronic organ rejection comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of or likely to chronically reject the transplant when the activity of the target protease in the sample
- the disclosure provides a method of monitoring chronic organ rejection in a transplant recipient subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of or likely to chronically reject the transplant when the activity of the target protease in the sample is greater than the reference activity
- Steps (a)-(g) can be repeated over a period of hours to months to years after the subject has received the transplanted tissue, for example, 3 hours to three weeks after receiving the transplanted tissue. In some embodiments, steps (a)-(g) are repeated every few months beginning one to thirty years after the subject has received the transplanted tissue. In other embodiments, steps (a)-(g) are repeated over a period of hours to weeks after the subject has clinical signs of graft functional decline, for example, 3 hours to three weeks after the first sign of graft functional decline.
- the disclosure provides a method of selecting a transplant recipient subject for a clinical trial for a chronic organ rejection therapeutic and/or prophylactic compositions and/or methods comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) suitable for the trial when the activity of the target protea
- the disclosure provides a method of treating a transplant recipient subject for chronic organ rejection comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of or likely to chronically reject the transplant when the activity of the target protease in the sample is greater than the reference activity
- the disclosure provides methods of diagnosing immune conditions related to T cell cytotoxicity (e.g., graft versus host disease (GvHD) and autoimmune diseases).
- the disclosure also provides methods of detecting such immune conditions in a patient, predicting such immune conditions in a patient, classifying a patient as having such immune conditions, monitoring such immune conditions in a patient, selecting a patient for a clinical trial for such immune condition-related therapeutic compositions and/or methods, and methods of treating such immune conditions in a patient.
- the detectable peptide sequences are released at the site of protease cleavage, producing a localized detectable signal (that can be detected at the site of cleavage or downstream of the site of cleavage), and can accumulate in draining lymph nodes, blood, urine, and other bodily fluids where they can be detected (e.g., by methods as described in US2014/0303014 and US2014/0363833, each of which is incorporated herein by reference).
- the disclosure provides a method of diagnosing immune conditions related to T cell cytotoxicity in a subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having the immune condition when the transplant when the activity of the target protease in the sample is greater than the reference activity of
- the disclosure provides a method of detecting immune conditions related to T cell cytotoxicity in a subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having the immune condition when the activity of the target protease in the sample is greater than the reference activity of the target prote
- the disclosure provides a method of predicting immune conditions related to T cell cytotoxicity in a subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) at risk of developing or likely to develop the immune condition when the activity of the target protease in the sample is greater than
- the disclosure provides a method of classifying a subject as having or likely to have immune conditions related to T cell cytotoxicity comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of developing, or likely to develop the immune condition when the activity of the target protea
- the disclosure provides a method of monitoring immune conditions related to T cell cytotoxicity in a subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of developing, or likely to develop the immune condition when the activity of the target protease in the sample is
- the disclosure provides a method of selecting a transplant recipient subject for a clinical trial for a therapeutic and/or prophylactic compositions and/or methods for a T-cell toxicity-related immune disorder comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) suitable for the
- the disclosure provides a method of treating a subject for a T-cell toxicity-related immune disorder comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of developing, or likely to develop the immune condition when the activity of the target protease in the
- the disclosure provides methods of diagnosing T cell cytotoxicity (e.g., to predict treatment efficacy in patients being treated with cancer immune therapies such as checkpoint blockade inhibitors or CAR T cell therapies).
- the disclosure also provides methods of detecting T cell cytotoxicity in a patient, predicting T cell cytotoxicity in a patient, classifying a patient as having T cell cytotoxicity, monitoring T cell cytotoxicity in a patient, selecting a patient for a clinical trial for T cell cytotoxicity-related therapeutic compositions and/or methods, and methods of treating T cell cytotoxicity in a patient.
- the detectable peptide sequences are released at the site of protease cleavage, producing a localized detectable signal (that can be detected at the site of cleavage or downstream of the site of cleavage), and can accumulate in draining lymph nodes, blood, urine, and other bodily fluids where they can be detected (e.g., by methods as described in US2014/0303014 and US2014/0363833, each of which is incorporated herein by reference).
- the disclosure provides a method of diagnosing T cell cytotoxicity in a subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of developing, or likely to develop T cell cytotoxicity when the activity of the target protease in the sample is greater
- the disclosure provides a method of detecting T cell cytotoxicity in a subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of developing, or likely to develop T cell cytotoxicity when the activity of the target protease in the sample is greater
- the disclosure provides a method of predicting T cell cytotoxicity in a subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of developing, or likely to develop T cell cytotoxicity when the activity of the target protease in the sample is greater
- the disclosure provides a method of classifying a subject as having or likely to have T cell cytotoxicity comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of developing, or likely to develop T cell cytotoxicity when the activity of the target protease
- the disclosure provides a method of monitoring T cell cytotoxicity in a subject comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i)) having, at risk of developing, or likely to develop T cell cytotoxicity when the activity of the target protease in the sample is greater
- the disclosure provides a method of selecting a subject for a clinical trial for T cell cytotoxicity-related therapeutic and/or prophylactic compositions and/or methods comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) suitable for the trial when the activity of the target protea
- the disclosure provides a method of treating a subject for T cell cytotoxicity comprising: (a) administering a composition comprising a nanosensor to the subject, the nanosensor comprising a scaffold; a linker coupled to the scaffold domain; at least one peptide substrate coupled to the linker, the peptide substrate comprising a target protease cleavage sequence; and a detectable reporter coupled to the peptide substrate; (b) obtaining a sample of a bodily fluid from the subject; (c) detecting a level of the detectable reporter in the sample of the bodily fluid; (d) determining an activity of the target protease based on the level of the detectable reporter in the sample of the bodily fluid; (e) comparing the activity of the target protease in the sample to a reference activity of the target protease; (f) identifying the subject as: (i) having, at risk of developing, or likely to develop T cell cytotoxicity when the activity of the target protease in the sample is greater than
- the composition comprising the nanosensor is administered intravenously.
- the composition comprising the nanosensor further comprises a pharmaceutically acceptable carrier and/or adjuvant.
- the peptide substrate comprises a detectable peptide sequence as discussed herein, e.g., a detectable peptide sequence comprising an amino acid sequence comprising one or more of SEQ ID NOs 2-136.
- the detectable reporter can be, for example and not limitation, a fluorophore, a mass spectrometry bar code, and/or an imaging agent (e.g., a contrast imaging agent or a PET imaging agent).
- the bodily fluid can be, for example and not limitation, urine, blood, lymphatic fluid, plasma, and/or saliva.
- a sample of a bodily fluid is obtained 30 minutes to 48 hours, including but not limited to 30 minutes to 6 hours, after administration of the composition comprising the nanosensor.
- the target protease is selected from the group consisting of Granzyme B, Granzyme A, MALT1, Caspase 8, Calpain 2, Cathepsin X, Cls, Clr, MASP2, Factor I, Factor D, ADAMTS1, MMP2, and MMP9.
- the reporter domain enables detection by light spectroscopy, near-infrared imaging, fluorescent imaging, bioluminescent imaging, ELISA, PCR (for DNA barcodes), spectrophotometry, mass spectrometry (e.g., bar code mass spectrometry), and/or imaging (e.g., CAT, MRI, PET), and/or by methods described in any of WO2007/106415, US2010/0240050, US2014/0303014 and US2014/0363833, each of which is incorporated herein by reference.
- a single sample of bodily fluid is taken from the subject for analysis.
- multiple samples of bodily fluid are taken in order to monitor the protease activity, and thus acute and/or chronic rejection, over time. Such samples can be taken over a period of 30 minutes to 4 weeks after the transplant occurs in order to monitor development of acute and/or chronic rejection.
- the composition comprising the nanosensor can be administered multiple times to the subject before each bodily fluid sample is taken for analysis.
- acute and/or chronic rejection can be monitored over the patient's lifetime after receiving the transplanted tissue, and thus may occur months to years to decades after receiving the transplanted tissue.
- the monitoring may be triggered by a clinical change or symptom development, such as clinical signs of graft function decline. In such embodiments, the monitoring may occur for hours to weeks to months after onset of the clinical change or symptom development.
- the reference sample is a sample taken from the same subject prior to the transplant, while in other embodiments the reference sample is a sample taken from a person of similar physical characteristics as the subject (e.g., BMI-, age-, and gender-matched person who is not undergoing a transplant, does not require transplant surgery, and/or does not have an immune disorder as determined by standard examination and diagnostic methods).
- the reference sample provides a control level of the target protease activity. If the subject is determined to be acutely and/or chronically rejecting the transplanted tissue, therapeutic and/or prophylactic compositions and/or methods can be employed to treat or prevent the acute and/or chronic rejection.
- Such therapeutic compositions and/or methods include, for example and not limitation, salvage therapy with thymoglobulin and steroids, immunosuppression therapies (such as for example and not limitation, anti-inflammatory drugs (cortisol, prednisone, dexamethasone, fludrocortisone acetate), anti-proliferatives (cyclophosphamide, methotrexate, azathioprine, mytomycin C)), and T cell targeted therapies (cyclosporine, tacrolimus, sirolimus, thymoglobulin, OKT3, antithymocyte globulin, Basiliximab, Belatacept, Abatacept).
- immunosuppression therapies such as for example and not limitation, anti-inflammatory drugs (cortisol, prednisone, dexamethasone, fludrocortisone acetate), anti-proliferatives (cyclophosphamide, methotrexate, azathioprine, mytomycin C
- compositions and/or methods can be employed to treat or prevent the immune condition, such as for example and not limitation, immunosuppression therapies (such as for example and not limitation, anti-inflammatory drugs (cortisol, prednisone, dexamethasone, fludrocortisone acetate), anti-proliferatives (cyclophosphamide, methotrexate, azathioprine, mytomycin C)), and T cell targeted therapies (cyclosporine, tacrolimus, sirolimus, thymoglobulin, OKT3, antithymocyte globulin, Basiliximab, Belatacept, Abatacept).
- immunosuppression therapies such as for example and not limitation, anti-inflammatory drugs (cortisol, prednisone, dexamethasone, fludrocortisone acetate), anti-proliferatives (cyclophosphamide, methotrexate, azathioprine, mytomycin C)
- steps (d) and/or (e) and/or (f) are performed by a computer, as described further herein.
- certain embodiments of the methods of the disclosure comprise a further step of preparing a report containing results from the identification, wherein said report is written in a computer readable medium, printed on paper, or displayed on a visual display.
- the method may further comprise treatment and/or prophylaxis of patients determined to have, or be at risk for having, acute or chronic rejection or immune conditions related to T cell cytotoxicity (e.g., autoimmune diseases, GVHD, etc).
- the method of treatment and/or prophylaxis may comprise detection of the biomarkers and their use in the described algorithm to identify patients having or being at risk of having severe or lethal GVHD, and initiating treatment and/or prophylaxis based on the identification, or being suitable for undergoing such therapies.
- the treatment and/or prophylaxis comprises administration of appropriate therapeutically effective pharmaceutical compositions and/or use of appropriate therapeutically effective methods.
- Non-limiting examples of the inflammatory and autoimmune diseases treatable by the methods of the present disclosure include, e.g., inflammatory bowel disease (IBD), graft versus host disease (GVHD), ulcerative colitis (UC), Crohn's disease, diabetes (e.g., diabetes mellitus type 1), multiple sclerosis, arthritis (e.g., rheumatoid arthritis), Graves' disease, lupus erythematosus, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), autoimmune myopathies, Sjogren's syndrome (SS), vasculitis, scleroderma, ankylosing spondylitis, psoriasis, Behcet's disease, autistic enterocolitis, Guillain-Barre Syndrome, myasthenia gravis, pemphigus vulgaris, acute disseminated encephalomyelitis (ADEM), transverse myelitis
- compositions and methods of the present disclosure can be combined with other therapeutic agents suitable for the same or similar diseases.
- two or more embodiments of the disclosure may be also co-administered to generate additive or synergistic effects.
- the embodiment of the disclosure and the second therapeutic agent may be simultaneously or sequentially (in any order). Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.
- the disclosure can be combined with other therapies that block inflammation (e.g., via blockage of ILL INF ⁇ / ⁇ , IL6, TNF, IL13, IL23, etc.).
- compositions and methods of the disclosure can be also administered in combination with an anti-tumor antibody or an antibody directed at a pathogenic antigen or allergen.
- compositions and methods of the disclosure can be combined with other immunomodulatory treatments such as, e.g., therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.) or activators (including but not limited to agents that enhance 41BB, OX40, etc.).
- therapeutic vaccines including but not limited to GVAX, DC-based vaccines, etc.
- checkpoint inhibitors including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.
- activators including but not limited to agents that enhance 41BB, OX40, etc.
- the inhibitory treatments of the disclosure can be also combined with other treatments that possess the ability to modulate NKT function or stability, including but not limited to CD1d, CD1d-fusion proteins, CD1d dimers or larger polymers of CD1d either unloaded or loaded with antigens, CD1d-chimeric antigen receptors (CD1d-CAR), or any other of the five known CD1 isomers existing in humans (CD1a, CD1b, CD1c, CD1e), in any of the aforementioned forms or formulations, alone or in combination with each other or other agents.
- CD1d CD1d-fusion proteins
- CD1d-chimeric antigen receptors CD1d-chimeric antigen receptors
- CD1d-CAR CD1d-chimeric antigen receptors
- Therapeutic methods of the disclosure can be combined with additional immunotherapies and therapies.
- the methods and compositions of the disclosure can be used in combination with conventional cancer therapies, such as, e.g., surgery, radiotherapy, chemotherapy or combinations thereof, depending on type of the tumor, patient condition, other health issues, and a variety of factors.
- other therapeutic agents useful for combination cancer therapy with the inhibitors of the disclosure include anti-angiogenic agents.
- anti-angiogenic agents include, e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000).
- the methods and compositions of the disclosure can be used in combination with a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab or ranibizumab).
- a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab or ranibizumab).
- compositions of the disclosure can comprise a carrier and/or excipient. While it is possible to use a compound of the present disclosure for therapy as is, it may be preferable to administer it in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient and/or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
- a suitable pharmaceutical excipient and/or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
- the excipient and/or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Acceptable excipients and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit. 2005).
- compositions readily accommodate additional mixtures, such as, e.g., milk, yogurt, and infant formula.
- Solid dosage forms for oral administration can also be used and can include, e.g., capsules, tablets, caplets, pills, troches, lozenges, powders, and granules.
- suitable excipients include, e.g., diluents, buffering agents (e.g., sodium bicarbonate, infant formula, sterilized human milk, or other agents which allow bacteria to survive and grow [e.g., survive in the acidic environment of the stomach and to grow in the intestinal environment]), preservatives, stabilizers, binders, compaction agents, lubricants, dispersion enhancers, disintegration agents, antioxidants, flavoring agents, sweeteners, and coloring agents.
- buffering agents e.g., sodium bicarbonate, infant formula, sterilized human milk, or other agents which allow bacteria to survive and grow [e.g., survive in the acidic environment of the stomach and to grow in the intestinal environment]
- preservatives e.g., sodium bicarbonate, infant formula, sterilized human milk, or other agents which allow bacteria to survive and grow [e.g., survive in the acidic environment of the stomach and to grow in the intestinal environment]
- preservatives e.g.
- the composition is formulated for delivery by a route such as, e.g., oral, topical, rectal, mucosal, sublingual, nasal, naso/oro-gastric gavage, parenteral, intraperitoneal, intravenous, intradermal, transdermal, intrathecal, nasal, and intracheal administration.
- the composition is in a form of a liquid, foam, cream, spray, powder, or gel.
- the composition comprises a buffering agent (e.g., sodium bicarbonate, infant formula or sterilized human milk).
- the composition is formulated for intravenous administration.
- Non-limiting examples of useful routes of delivery include oral, rectal, fecal (by enema), and via naso/oro-gastric gavage, as well as parenteral, intraperitoneal, intravenous, intradermal, transdermal, intrathecal, nasal, and intracheal administration.
- the active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.
- the formulation can include added ingredients to improve palatability, improve shelf-life, improve absorption, impart nutritional benefits, and the like.
- the useful dosages of the compounds and formulations of the disclosure can vary widely, depending upon the nature of the disease, the patient's medical history, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like.
- the initial dose may be larger, followed by smaller maintenance doses.
- the dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level. It is contemplated that a variety of doses can be effective to achieve a therapeutic effect.
- a compound of the present disclosure for therapy as is, it may be preferable to administer it in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
- a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
- the excipient, diluent and/or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
- Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit. 2005).
- oral delivery is preferred for delivery to the digestive tract because of its ease and convenience, and because oral formulations readily accommodate additional mixtures, such as milk, yogurt, and infant formula.
- Oral delivery may also include the use of nanoparticles that can be targeted, e.g., to the GI tract of the subject, such as those described in Yun et al., Adv Drug Deliv Rev. 2013, 65(6):822-832 (e.g., mucoadhesive nanoparticles, negatively charged carboxyl ate- or sulfate-modified particles, etc.).
- nanoparticles that can be targeted, e.g., to the GI tract of the subject, such as those described in Yun et al., Adv Drug Deliv Rev. 2013, 65(6):822-832 (e.g., mucoadhesive nanoparticles, negatively charged carboxyl ate- or sulfate-modified particles, etc.).
- Non-limiting examples of other methods of targeting delivery of compositions to the GI tract are discussed in U.S. Pat. Appl. Pub. No.
- pH sensitive compositions such as, e.g., enteric polymers which release their contents when the pH becomes alkaline after the enteric polymers pass through the stomach
- compositions for delaying the release e.g., compositions which use hydrogel as a shell or a material which coats the active substance with, e.g., in vivo degradable polymers, gradually hydrolyzable polymers, gradually water-soluble polymers, and/or enzyme degradable polymers]
- bioadhesive compositions which specifically adhere to the colonic mucosal membrane, compositions into which a protease inhibitor is incorporated, a carrier system being specifically decomposed by an enzyme present in the colon).
- Oral pharmaceutical dosage forms include, by way of example and without limitation, solid, gel and liquid.
- Solid dosage forms include tablets, capsules, granules, and bulk powders.
- Oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated.
- Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.
- the active ingredient(s) can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
- the active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.
- inactive ingredients examples include red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink.
- Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract.
- Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
- the formulations are solid dosage forms, such as capsules or tablets.
- the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or agents of a similar nature: a binder; a diluent; a disintegrating agent; a lubricant; a glidant; a sweetening agent; and a flavoring agent.
- the agent could be provided in a composition that protects it from the acidic environment of the stomach.
- the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active agent in the intestine.
- the composition may also be formulated in combination with an antacid or other such ingredient.
- dosage unit form When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as fatty oil.
- dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
- the agents can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like.
- a syrup may contain, in addition to the active agents, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
- the active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics.
- Pharmaceutically acceptable carriers included in tablets are binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, and wetting agents.
- Enteric-coated tablets because of the enteric-coating, resist the action of stomach acid and dissolve or disintegrate in the neutral or alkaline intestines.
- Sugar-coated tablets are compressed tablets to which different layers of pharmaceutically acceptable substances are applied.
- Film-coated tablets are compressed tablets which have been coated with a polymer or other suitable coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle utilizing the pharmaceutically acceptable substances previously mentioned.
- Coloring agents may also be used in the above dosage forms.
- Flavoring and sweetening agents are used in compressed tablets, sugar-coated, multiple compressed and chewable tablets. Flavoring and sweetening agents are useful in the formation of chewable tablets and lozenges.
- Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
- Aqueous solutions include, for example, elixirs and syrups.
- Emulsions are either oil-in-water or water-in-oil.
- Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
- Solutions or suspensions can include any of the following components, in any combination: a sterile diluent, including by way of example without limitation, water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. In instances in which the agents exhibit insufficient solubility, methods for solubilizing agents may be used.
- a sterile diluent including by way of example without limitation, water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent
- antimicrobial agents such as benz
- Such methods include, but are not limited to, using co-solvents, such as, e.g., dimethylsulfoxide (DMSO), using surfactants, such as TWEEN® 80, or dissolution in aqueous sodium bicarbonate.
- co-solvents such as, e.g., dimethylsulfoxide (DMSO)
- surfactants such as TWEEN® 80
- dissolution in aqueous sodium bicarbonate such as sodium bicarbonate.
- Pharmaceutically acceptable derivatives of the agents may also be used in formulating effective pharmaceutical compositions.
- the composition can contain along with the active agent, for example and without limitation: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acacia gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art.
- a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose
- a lubricant such as magnesium stearate, calcium stearate and talc
- a binder such as starch, natural gums, such as gum acacia gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone
- Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active agent as defined above and optional pharmaceutical adjuvants in a carrier, such as, by way of example and without limitation, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
- a carrier such as, by way of example and without limitation, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
- the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, such as, by way of example and without limitation, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
- auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, such as, by way of example and without limitation, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
- auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, such as, by way of example and without limitation, acetate, sodium citrate, cyclodextr
- the active agents or pharmaceutically acceptable derivatives may be prepared with carriers that protect the agent against rapid elimination from the body, such as time release formulations or coatings.
- the compositions may include other active agents to obtain desired combinations of properties.
- Parenteral administration generally characterized by injection, either subcutaneously, intramuscularly or intravenously, is also contemplated herein.
- Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
- Suitable excipients include, by way of example and without limitation, water, saline, dextrose, glycerol or ethanol.
- compositions to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
- auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
- an inhibitor of Nt5e or A1R is dispersed in a solid inner matrix (e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl acetate) that is surrounded by an outer polymeric membrane (e.g., polyethylene,
- Lyophilized powders can be reconstituted for administration as solutions, emulsions, and other mixtures or formulated as solids or gels.
- the sterile, lyophilized powder is prepared by dissolving an agent provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent.
- the solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder.
- Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
- the solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, typically, about neutral pH.
- a buffer such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, typically, about neutral pH.
- sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation.
- the resulting solution can be apportioned into vials for lyophilization.
- Each vial can contain, by way of example and without limitation, a single dosage (10-1000 mg, such as 100-500 mg) or multiple dosages of the agent.
- the lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration.
- about 1-50 mg, such as about 5-35 mg, for example, about 9-30 mg of lyophilized powder is added per
- composition or pharmaceutically acceptable derivatives thereof may be formulated as aerosols for application, e.g., by inhalation or intranasally (e.g., as described in U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923).
- These formulations can be in the form of an aerosol or solution for a nebulizer, or as a microtine powder for insufflation, alone or in combination with an inert carrier such as lactose.
- the particles of the formulation can, by way of example and without limitation, have diameters of less than about 50 microns, such as less than about 10 microns.
- the agents may be also formulated for local or topical application, such as for application to the skin and mucous membranes (e.g., intranasally), in the form of nasal solutions, gels, creams, and lotions.
- local or topical application such as for application to the skin and mucous membranes (e.g., intranasally), in the form of nasal solutions, gels, creams, and lotions.
- Transdermal patches including iontophoretic and electrophoretic devices, are well known to those of skill in the art. For example, such patches are disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.
- kits useful in the practice of the methods of the invention comprise detection reagents that specifically bind the nanosensors of the present invention, such as for example and not limitation, at least one of the detectable peptide sequences.
- the kits typically include a probe that comprises an antibody or nucleic acid sequence that specifically binds to polypeptides or polynucleotides of the invention, such as for example and not limitation, at least one of the detectable peptide sequences comprising the amino acid sequence of SEQ ID NOs 2-136, as well as genes encoding these sequences, and a label for detecting the presence of the probe.
- kits may include several antibodies specific for, or polynucleotide sequences encoding, the polypeptides of the invention.
- the kits may further comprise control probes for detection of a control nucleic acid or a control protein in order to provide a control level of the nucleic acid or protein, and/or other standards or controls.
- the probe is optionally detectably labeled.
- the kit may contain in separate containers a nucleic acid or antibody (either already bound to a solid matrix or packaged separately with reagents for binding them to the matrix), control formulations (positive and/or negative), and/or a detectable label such as fluorescein, green fluorescent protein, rhodamine, cyanine dyes, Alexa dyes, luciferase, radiolabels, among others. Instructions for carrying out the assay may also be included in the kit.
- the assay may, for example and not limitation, be in the form of a Northern hybridization, sandwich ELISA or protein antibody array.
- Reagents for detecting biomarkers of the present invention can be immobilized on a solid matrix such as a porous strip to form at least one detectable peptide sequence detection site.
- the measurement or detection region of the porous strip may include a plurality of sites containing an antibody or nucleic acid.
- a test strip may also contain sites for negative and/or positive controls. Alternatively, control sites can be located on a separate strip from the test strip.
- the different detection sites may contain different amounts of immobilized antibodies or nucleic acids, e.g., a higher amount in the first detection site and lesser amounts in subsequent sites.
- the number of sites displaying a detectable signal provides a quantitative indication of the amount of detectable peptide sequence present in the sample.
- the detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
- the kit contains a nucleic acid substrate array comprising one or more nucleic acid sequences.
- the nucleic acids on the array specifically identify one or more nucleic acid sequences adapted to bind a nucleic acid sequence encoding a detectable peptide sequence comprising at least one of SEQ ID NOs 2-136.
- the substrate array can be on, e.g., a solid substrate or “chip”. Alternatively, the substrate array can be a solution array.
- kits of the invention can also provide reagents for primer extension and amplification reactions.
- the kit may further include one or more of the following components: a reverse transcriptase enzyme, a DNA polymerase enzyme (such as, e.g., a thermostable DNA polymerase), a polymerase chain reaction buffer, a reverse transcription buffer, and deoxynucleoside triphosphates (dNTPs).
- a reverse transcriptase enzyme such as, e.g., a thermostable DNA polymerase
- a polymerase chain reaction buffer such as, e.g., a thermostable DNA polymerase
- dNTPs deoxynucleoside triphosphates
- a kit can include reagents for performing a hybridization assay for nucleic acid(s) and/or proteins.
- the detecting agents can include nucleotide analogs and/or a labeling moiety, e.g., directly detectable moiety such as a fluorophore (fluorochrome) or a radioactive isotope, or indirectly detectable moiety, such as a member of a binding pair, such as biotin, or an enzyme capable of catalyzing a non-soluble colorimetric or luminometric reaction.
- the kit may further include at least one container containing reagents for detection of electrophoresed nucleic acids.
- Such reagents include those which directly detect nucleic acids, such as fluorescent intercalating agent or silver staining reagents, or those reagents directed at detecting labeled nucleic acids, such as, but not limited to, ECL reagents.
- a kit can further include DNA or RNA isolation or purification means as well as positive and negative controls.
- the kit may include at least one container containing reagents for detection of electrophoresed proteins.
- Such reagents include those which directly detect proteins, such as Coomassie blue or other staining reagents including fluorescent staining agents, or those reagents directed at detecting labeled proteins.
- a kit can further include protein isolation or purification means as well as positive and negative controls.
- a kit can also include a notice associated therewith in a form prescribed by a governmental agency regulating the manufacture, use or sale of diagnostic kits. Detailed instructions for use, storage and trouble-shooting may also be provided with the kit.
- a kit can also be optionally provided in a suitable housing that is preferably useful for robotic handling in a high throughput setting.
- the components of the kit may be provided as dried powder(s).
- the powder can be reconstituted by the addition of a suitable solvent.
- the solvent may also be provided in another container.
- the container will generally include at least one vial, test tube, flask, bottle, syringe, and/or other container means, into which the solvent is placed, optionally aliquoted.
- the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other solvent.
- the kit also will generally contain a second, third, or other additional container into which the additional components may be separately placed.
- additional components may be separately placed.
- various combinations of components may be comprised in a container.
- kits may also include components that preserve or maintain DNA or RNA, such as reagents that protect against nucleic acid degradation. Such components may be nuclease or RNase-free or protect against RNases, for example. Such kits may also include components that preserve or maintain proteins, such as reagents that protect against protein degradation. Any of the compositions or reagents described herein may be components in a kit.
- the kit further comprises an apparatus for collecting a bodily fluid sample, e.g., a urine, blood, lymphatic fluid, saliva and/or plasma sample, from a subject.
- a bodily fluid sample e.g., a urine, blood, lymphatic fluid, saliva and/or plasma sample
- the kit further comprises instructions for using the collection apparatus and/or the reagents comprising the kit.
- the methods and information described herein may be implemented, in all or in part, as computer executable instructions on known computer readable media.
- the methods described herein may be implemented in hardware.
- the method may be implemented in software stored in, for example, one or more memories or other computer readable medium and implemented on one or more processors.
- the processors may be associated with one or more Controllers, calculation units and/or other units of a computer system, or implanted in firmware as desired.
- the routines may be stored in any computer readable memory such as in RAM, ROM, flash memory, a magnetic disk, a laser disk, or other storage medium, as is also known.
- this software may be delivered to a computing device via any known delivery method including, for example, over a communication channel such as a telephone line, the Internet, a wireless connection, etc., or via a transportable medium, such as a computer readable disk, flash drive, and the like.
- a communication channel such as a telephone line, the Internet, a wireless connection, etc.
- a transportable medium such as a computer readable disk, flash drive, and the like.
- the various steps described above may be implemented as various blocks, operations, tools, modules and techniques which, in turn, may be implemented in hardware, firmware, software, or any combination of hardware, firmware, and/or software.
- some or all of the blocks, operations, techniques, etc. may be implemented in, for example, a custom integrated circuit (IC), an application specific integrated circuit (ASIC), a field programmable logic array (FPGA), a programmable logic array (PLA), etc.
- the software When implemented in software, the software may be stored in any known computer readable medium such as on a magnetic disk, an optical disk, or other storage medium, in a RAM or ROM or flash memory of a computer, processor, hard disk drive, optical disk drive, tape drive, etc. Likewise, the software may be delivered to a user or a computing system via any known delivery method including, for example, on a computer readable disk or other transportable computer storage mechanism.
- a system that is capable of carrying out a part or all of a method of the disclosure, or carrying out a variation of a method of the disclosure as described herein in greater detail.
- Exemplary systems include, as one or more components, computing systems, environments, and/or configurations that may be suitable for use with the methods and include, but are not limited to, personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, and the like.
- a system of the disclosure includes one or more machines used for analysis of biological material (e.g., genetic material), as described herein. In some variations, this analysis of the biological material involves a chemical analysis and/or a nucleic acid amplification.
- the computer may operate in a networked environment using logical connections to one or more remote computers, such as a remote computer via a network interface controller (NIC).
- the remote computer may be a personal computer, a server, a router, a network PC, a peer device or other common network node, and typically includes many or all of the elements described above relative to the computer.
- the logical connection between the NIC and the remote computer may include a local area network (LAN), a wide area network (WAN), or both, but may also include other networks.
- LAN local area network
- WAN wide area network
- the remote computer may also represent a web server supporting interactive sessions with the computer; or in the specific case of location-based applications may be a location server or an application server.
- the network interface may use a modem when a broadband connection is not available or is not used. It will be appreciated that the network connection shown is exemplary and other means of establishing a communications link between the computers may be used.
- the synthetic biomarkers consisted of a nanoparticle core conjugated with peptide substrates specific for Granzyme B (GzmB) ( FIG. 2A ).
- GzmB Granzyme B
- the use of a nanoparticle chaperone achieved two goals: it extended the half-life of surface-conjugated peptides by preventing renal clearance, and through passive targeting, accumulated in tissues with fenestrated endothelium including secondary lymphoid organs (e.g., spleen, lymph nodes) as well as sites of inflammation as occurs during graft rejection (22, 23).
- the second component of the synthetic biomarkers was a peptide substrate designed to sense a target protease of interest, which in this case is GzmB, the key cytotoxic effector protease secreted by CD8 T cells (25). These substrates were further modified with a reporter which allows detection of cleaved peptide fragments in urine.
- peptide substrates can be labelled with different barcoded technologies such as isobaric mass tags for multiplexing analysis, ligand encoded reporters for point of care paper-based tests (17-20).
- the inventors decorated nanoparticle surface with polyethylene glycol (PEG), which reduces nanoparticle uptake by the reticuloendothelial system (RES) (26).
- Hydrodynamic size profiling by dynamic light scattering (DLS) showed that PEGylated nanoparticles were stable in both PBS and mouse plasma, with the Z-average in plasma was 47 nm ( FIG. 7A ).
- the circulation half-life of synthetic biomarkers was ⁇ 3 hours in mice, which was consistent with previously reported values for clinically approved IONPs ( FIG. 7B ) (27).
- the inventors first set out to identify peptide substrates that are sensitive to cleavage by recombinant GzmB. From published literature (28-31), 13 candidate substrates ( FIG. 6 ) were pooled, each spanning 6-8 amino acids in length and containing conserved residues isoleucine at position P4 and aspartic acid at P1—the position immediately N-terminal of the cleavage site. The inventors varied the amino acids at other locations, length of the substrate, and flexible spacers on both sides of the designed substrate.
- the inventors identified the sequence AIEFD
- Michaelis-Menten kinetics analyses were performed to assess the efficiency of GzmB cleavage of substrate on nanoparticle surface. Fitted k cat /K M was 1.09 ⁇ 10 4 M ⁇ 1 s ⁇ 1 , similar to reported values of GzmB cleaving free substrates of similar sequences ( FIG. 2B ) (28, 31), which shows that presentation of substrates on nanoparticle surface does not affect GzmB cleavage.
- the coagulation and complement cascades contain ubiquitous circulating proteases that can degrade the synthetic biomarkers. Therefore, to test specificity of the substrate for GzmB, the inventors studied synthetic biomarker cleavage by key recombinant proteases as well as activated coagulation and complement cascades in blood samples. Increases in fluorescent intensity were observed only in samples containing recombinant GzmB ( FIGS. 2C, 2D, and 2E ). Furthermore, the formation of membrane attack complex (MAC) was not detected when synthetic biomarkers were incubated with serum samples, indicating that the synthetic biomarkers did not activate the complement cascade via interaction with foreign surface (32) ( FIG. 2F ). Overall, these data showed that synthetic biomarkers are specific for GzmB and are not activated by proteases in the coagulation and complement cascades.
- MAC membrane attack complex
- GzmB is the central effector protease that CD8 T cells utilize to kill target cells.
- GzmB is upregulated and contained in cytolytic granules, and upon TCR-pMHC engagement, these granules are lysed to release GzmB that enters target cells through perforin-mediated pore formation (25).
- GzmB serves as potent initiator of the apoptosis cascade by either direct cleavage to activate proapoptotic protease Caspase 3 or induction of mitochondrial disruption (25, 33).
- elevated levels of GzmB in blood has been used to monitor the efficacy of CAR T cell therapy against B cell leukemia (34).
- GzmB expression in longitudinal tumor biopsies could be used to differentiate non-responders from responders to immune blockade therapies (35).
- transplant rejection presence of GzmB mRNA transcript in patient urine samples has been shown to correlate to episodes of acute rejection (13, 14).
- transgenic OT-1 T cells which recognize the peptide epitope SIINFEKL from chicken ovalbumin (OVA), in a T cell killing assay (36).
- OVA ovalbumin
- the inventors performed flow analysis and confirmed that GzmB levels were elevated in OT-1 T cells after engagement with EG7-OVA target cells compared to EL4 controls ( FIGS. 8A-8B ).
- a fluorogenic assay in which a substrate can fluoresce upon cleavage by active GzmB was used to measure GzmB activity inside target cells ( FIG.
- the synthetic biomarkers amplified cytotoxic signal from transgenic OT1 T cells killing EG7-OVA target cells ( FIGS. 3E, 3F ). Furthermore, the initial velocity of probe activation was strongly correlated to the amount of secretory GzmB in coculture supernatant ( FIG. 3D, 3G ). After showing that the synthetic biomarkers could sense transgenic T cell killing with only one unique TCR-pMHC interaction, the inventors investigated their ability to sense alloreactive T cell killing, which is a polyclonal T cell response against alloantigens.
- splenocytes from donor BALB/c mice were co-incubated with splenocytes and lymphocytes from recipient C57BL/6 mice bearing skin grafts before adding synthetic biomarkers to monitor cytotoxic activity ( FIG. 3H ).
- Increased activation kinetics of synthetic biomarkers in cocultures containing T cells from mice bearing allografts was detected ( FIG. 3I ).
- the engineered GzmB-sensing synthetic biomarkers are useful for detection of alloreactive T cell killing.
- GzmB is Upregulated at the Onset of Acute Cellular Rejection
- host CD8 T cells activate through direct or indirect pathways by interaction with donor- or host-derived antigen presenting cells (APC) respectively, expand rapidly in secondary lymphoid organs (e.g., spleen and lymph nodes), and secrete cytotoxic granules containing GzmB to mediate allograft rejection (3, 4).
- ACR donor- or host-derived antigen presenting cells
- the core biopsy is used to examine for histological hallmarks of ACR—which directly result from T cell activity—including T cell infiltration, inflammatory cytokines, morphological changes, tissue remodeling (6).
- these morphological biomarkers which are downstream and lack predictive value, the ability to detect proteases that drive disease pathology can allow anticipation of patient trajectory, such as monitoring MMP activity in liver fibrosis progression and regression (17-21).
- the inventors sought to validate the expression of GzmB at the onset of acute rejection using the well-established BALB/c to B6 mouse model of skin transplantation ( FIG. 4A ).
- the inventors assigned a score of 4 for healthy grafts, a score of 0 for rejected grafts, and intermediate scores based on features such as the ratio of viable to necrotic skin and the presence of ulcerations or scabs.
- Allograft scores began to decrease significantly compared to isograft controls (2.6 vs. 3.9, P ⁇ 0.0001; FIGS. 4B, 4C and FIG. 9 ) and reached an endpoint when grafts were completely rejected two weeks post-transplant.
- the inventors stained skin grafts and tissue sections and found significant upregulation of CD8 and GzmB in skin allografts ( FIG. 4D , FIGS. 10A, 10B ). By contrast, GzmB was not detected in plasma samples throughout the course of rejection ( FIG. 4E ).
- the inventors analyzed CD8 T cells in splenocytes and lymphocytes from mice bearing skin grafts before and during the peak of rejection (day 5 to day 9).
- DAMPS damaged associated molecular patterns
- the inventors intravenously administered surface-labelled nanoparticles to C57BL/6 mice transplanted with both skin allografts and isografts on the same recipient at the onset of rejection (day 7) to allow quantification by full-body fluorescent imaging ( FIG. 5A ). Whereas both skin graft scores were statistically identical ( FIG. 4C ), the inventors found a 6-fold higher accumulation of nanoparticles in allografts compared to isografts or healthy skin (***P, n 3) ( FIGS. 5B, 5C , and FIG. 11 ).
- Pre-graft urine signals served as an internal baseline for each mouse to control for urine clearance rate and confounding variables associated with transplant surgery.
- the inventors did not detect significant elevation in post-graft urine signals, with respect to pre-graft signals, from na ⁇ ve mice, isograft mice, and allograft mice depleted of CD8 T cells.
- the core biopsy is considered the “gold” standard for diagnosing anti-graft activity.
- the biopsy is associated with significant patient morbidity and sampling error.
- activity-based nanosensors consisting of a nanoparticle core decorated with peptide substrates to sense the proteolytic activity of GzmB during antigen-specific CD8 T cell killing.
- synthetic biomarkers passively accumulate in allograft tissue during T-cell mediated rejection where they are cleaved by GzmB secreted by alloreactive T cells. These cleavage events trigger a pharmacokinetic switch where the cleaved peptide fragments, due to their small size, are filtered into urine to produce a noninvasive signal that predicts the onset of ACR.
- nanoparticle chaperon increases circulating half-life of substrate peptides which otherwise are cleared from the body by renal filtration within minutes after IV administration.
- This enhanced pharmacokinetics enables passive delivery of peptides to inflamed allograft tissues, which are active areas of T-cell mediated acute rejection.
- passive accumulation of synthetic biomarkers is significant in skin allograft, which is a thin and small piece of tissue, significantly higher accumulation would occur in allografts of larger and more solid organs.
- the inventors used IONPs as the carrier of substrate peptides because they are FDA-approved and have great translation potential.
- substrate peptides can be coated on other nanoscale scaffolds (e.g., PEG, protein-based carriers, PLGA) to tune their pharmacokinetics and presentation to optimize urine signals. While upregulation of GzmB has been detected in patients with acute rejection episodes, this expression is mostly localized to disease tissues, making in vitro diagnosis with a simple blood draw especially challenging.
- nanoscale scaffolds e.g., PEG, protein-based carriers, PLGA
- the invented synthetic biomarkers monitor in vivo GzmB activity at sites of active rejection and offer two potential methods of amplifying detection signals: (1) enzymatic turnovers allows one endogenous copy of GzmB to cleave thousands of synthetic substrate peptides while (2) renal clearance allows concentration of this disease signal in a small urine volume.
- the inventors have developed a sensitive probe to detect the activity of GzmB, which is the key effector protease during T cell cytotoxicity.
- Previous studies have found a correlation between elevations of GzmB RNA transcripts in patient urine samples to acute allograft rejection episodes (13, 14). Instead of looking at GzmB abundance that does not differentiate between Serpin-bound (inactive) from active form of GzmB (15, 40), these synthetic biomarkers sense proteolytic activity of GzmB as a predictor for acute cellular rejection.
- a single biomarker reflective of a fundamental cellular process or mechanism can be broadly applied to detect or monitor a range of disease conditions.
- FDG-PET fluorodeoxyglucose positron-emission tomography
- synthetic biomarkers that monitor GzmB activity can be used in the diagnosis of immune conditions related to T cell cytotoxicity which include graft versus host disease (GvHD), autoimmune diseases, and immuno-oncology.
- the sensitivity of a single biomarker is dependent on the setting in which it is recommended for clinical use. For example, monitoring the levels of a single biomarker over time in high-risk patients can significantly increase detection sensitivity.
- PSA prostate-specific antigen
- PSA lacks specificity and sensitivity for general population screening; however, it is an excellent biomarker when used in high-risk patients such as monitoring recurrent after radiation therapies (44).
- this platform can be readily expanded into a multiplex probe set using mass barcodes (17) to obtain a protease signature that reflects a more complete picture of patient pathology.
- Multiplexing analysis improves diagnostic specificity by simultaneous monitoring the activities of disease-associated proteases on the background of T cell mediated cytotoxicity. This capability can be valuable in differentiating acute organ rejection from conditions such bacterial infections, viral infections, and cancers, which all involve T cell cytotoxicity but are also driven by unique protease subsets (bacterial proteases, viral proteases, and tumor-derived proteases) (45-47).
- Aminated IONPs were synthesized in house per published protocol. GzmB-sensing peptides were synthesized by Tufts University Core Facility peptide synthesis service. Aminated IONPs were first reacted to the biofunctional crosslinker Succinimidyl Iodoacetate (SIA; Thermo) for 2 hours at room temperature (RT) and excess SIA were removed by buffer exchange using Amicon spin filter (30 kDa, Millipore).
- SIA Succinimidyl Iodoacetate
- DLS measurements of synthetic biomarkers were done in PBS or mouse plasma at RT using Zetasizer Nano ZS (Malvern).
- the inventors administered via IV 20 ug of labelled IONPs to CFW Mice (Charles River).
- blood was collected into heparin-coated Capillary Tubes (VWR) via retro-orbital collection and imaged using Odyssey CLx Imaging System (LI-COR).
- Synthetic biomarkers (6 nM by NP, 300 nM by peptide) were incubated in PBS+1% bovine serum albumin (BSA; Sigma) at 37° C. with murine Granzyme B (0.2 ⁇ M, Peprotech), human thrombin (HaemTech), mouse thrombin (HaemTech), mouse plasmin (HaemTech), Clr (Sigma), Cls (Sigma), Factor D (Sigma), Factor I (Sigma), MASP2 (Biomatik). Sample fluorescence were measured for 60 minutes using Cytation 5 plate reader (Biotek).
- a library of potential substrates was synthesized by Tufts University Core Facility peptide synthesis service and conjugated to IONPs. Cleavage assays of nanoparticles decorated with these substrates with recombinant GzmB were performed, and data was fitted to compare initial cleavage velocities. To determine Michaelis-Menten constants, cleavage assays with GzmB were performed at different substrate concentrations. To initiate coagulation cascade, citrated plasma was mixed with synthetic biomarkers before addition of calcium chloride (15 mM, Sigma).
- Control Human Serum (Sigma) was mixed with synthetic biomarkers before addition of Heat Aggregated Gamma Globulin (HAGG; Quidel) per the manufacturer's protocol. After measuring fluorescence for 1 hour, supernatants were collected and measured for formation of MAC complex using MicroVue CH50 Eq EIA Kit (Quidel).
- HAGG Heat Aggregated Gamma Globulin
- EL4 and EG7-OVA cells were grown in RPMI 1640 supplemented with 10% FBS and 25 mM HEPES (Gibco). EG7-OVA cultures were supplemented with G418 (0.4 mg/ml, InvitroGen).
- CD8 T cells were isolated from OT1 (Jackson Labs) splenocytes by MACS using CD8a Microbeads (Miltenyi).
- Cells were activated by seeding in 96-well plates coated with anti-mouse CD3e (Clone: 145-2C11, BD) and anti-mouse CD28 (Clone: 37.51, BD) at 2 ⁇ 10 6 cells/ml in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin-streptomycin, 1 ⁇ non-essential amino acids (Gibco), 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, and 30 U/ml hIL-2 (Roche). After 2 days, cells were transferred to uncoated plates.
- CD8+ OT1 T cells were isolated and activated per above protocol. On day 5 post activation, 1 ⁇ 10 6 OT1 T cells were coincubated with 1 ⁇ 106 EL4 or EG7-OVA target cells for 2 hours at 37° C. Coculture supernatants were mixed with synthetic biomarkers (2 nM by NP, 100 nM by peptides) and fluorescence were monitored for 1 hour at 37° C. To sense alloreactive T cell killing, on day 7 post-transplant, CD8 T cells were isolated from splenocytes and lymphocytes of skin graft mice.
- Skin grafts were qualitatively scored on a scale ranging from 0-4 per established protocol by the Emory Transplant Center. Scoring involved direct observation and palpation of the graft and surrounding tissue. A score of 0 was characterized by complete tissue necrosis, stiffness, and severe discoloration. A score of 4 was characterized by incorporation with surrounding tissue, pliability, and healthy pigmentation.
- tissues were collected from skin graft mice at day 7 post-transplant. All tissues were fixed in 4% paraformaldehyde (EMS) overnight at 4° C., washed with PBS and stored in 70% ethanol (VWR) until paraffin-embedding, sectioning, and staining for GzmB and CD8 (Winship Pathology Core).
- EMS paraformaldehyde
- VWR 70% ethanol
- splenocytes or lymphocytes from skin graft mice were restimulated with 1 ⁇ 10 6 BALB/c splenocytes for 6 hours at 37° C. before staining for GzmB using anti-mouse GzmB (Clone: NGZB, eBioScience) and Intracellular Fixation & Permeabilization Buffer Set (eBioScience).
- mice On day 7 post-transplant, skin graft mice were administered with either NPs (20 ug) or peptides (10 nmol) labelled with VivoTag S-750 (VT750; PerkinElmer).
- VT750 VivoTag S-750
- IVIS Spectrum CT Imaging System PerkinElmer
- L-COR Odyssey CLx Imaging System
- IVIS Spectrum CT Imaging System PerkinElmer
- VT750-labelled synthetic biomarkers (10 nmol by peptides) were administered to skin graft mice.
- Major organs were excised and imaged with Odyssey CLx Imaging System (LI-COR) while mouse bladders were imaged with IVIS Spectrum CT Imaging System (PerkinElmer) after 90 minutes. All urinalysis experiments were done in paired setup. 4 days before and 7 days after surgeries, skin graft mice were administered with FITC-labelled Synthetic biomarkers (10 nmol by peptides). Urine were collected after 90 minutes by placing mice over 96-well plates.
- LI-COR Odyssey CLx Imaging System
- IVIS Spectrum CT Imaging System PerkinElmer
- FITC in urine was purified by a magnetic separation assay using Dynabeads (Thermo, 65501) coated with anti-FITC (GeneTex, GTX10257). Fluorescent signals were measured with Cytation 5 Plate Reader (Biotek). Concentrations of FITC were calculated using a free FITC ladder and normalized with urine volume. For CD8 depletion study, mice were given anti-mouse CD8 (clone: 53-6.7, BioXCell) for 3 consecutive days following with booster shots every 3 days after.
- Flow cytometry analysis of splenocytes and lymphocytes were performed with anti-mouse CD3 (clone: 17A2, Biolegend), anti-mouse CD4 (clone: RM4-5, Biolegend), anti-mouse CD8 (clone: KT15, Serotec) to confirm success of depletion.
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