Granzyme B detection

Abstract

The present disclosure relates to the development of novel cleavable peptide probes for use in the detection of Granzyme B, and methods of using the probes in medical and biological environments. The probes can include a fluorescent moiety linked or attached to a peptide, and a quenching moiety linked or attached to the peptide, where upon cleavage of the peptide, the fluorescent moiety emits light.

Description

[Technical field]

[0001] The present disclosure relates to the development of novel probes for use in the detection of Granzyme B and methods of using the probes in medical and biological settings. [Background technology]

[0002] CD8+ T cell responses are one of the major immune mechanisms that protect the human body against cancer. The presence of CD8+ T cells in tumors indicates a good prognosis in cancer patients. However, there is a large variability in how patients respond to immunotherapy. Although clinical imaging can measure tumor size and, to some extent, CD8+ T cell infiltration, it does not provide a readout of how efficiently the immune system responds to cancer cells. This limitation hinders the evaluation of new drugs and the personalized optimization of immunotherapy to minimize off-target toxicity. A chemical approach to directly measure the activity of tumor-infiltrating T cells would provide a reliable indicator of response to treatment and accelerate the screening of anticancer drugs.

[0003] Standard methods for in vitro monitoring of CD8+ T cells include cytotoxic LDH, MTS or 51 Cr release assays are used. These assays report bulk cytotoxicity rather than T cell specific anticancer responses. CD8+ T cell activity can be indirectly monitored by measuring the concentrations of extracellular cytokines and membrane proteins (e.g., CD107a) using antibodies. Although these methods assess T cell function, they do not directly report cancer cell death and are therefore not biomarkers of immune killing capacity in tumors. The chemical design of activity-sensing reporters of granzyme B (GzmB) is an effective strategy to monitor CD8+ T cell cytotoxic activity in cancer. GzmB is a serine protease that is stored inactively in T cells until recognition by an antigen prompts its release and activation in cancer cells. Probes for detecting GzmB include antibodies that do not distinguish between activated and inactivated forms of the enzyme. 1and fusion proteins, as well as activatable constructs based on the Ile-Glu-Pro-Asp (IEPD) sequence first described by Thornberry et al. 2 Some examples of the latter include nanoprobes for urine analysis. 3,4 All of these constructs have limited catalytic efficiency (e.g., pmol min -1 V in the range max and mM -1 s -1 Nearby k cat / K M The ratio depends on the chemical scaffold (see Table 1). It is an object of the present disclosure to avoid and / or mitigate one or more of the above mentioned disadvantages. Summary of the Invention

[0004] The present disclosure is based in part on the development of a probe that is cleavable by granzyme B and that upon cleavage produces a cleaved peptide that is detectable, for example, by an increased signal, a change in one or more physicochemical properties, or an increased signal-to-noise ratio.

[0005] In a first embodiment, a probe for use in detecting granzyme B is provided, the probe comprising a granzyme B cleavable peptide linked or conjugated to a detectable moiety, the granzyme B cleavable peptide comprising, consisting essentially of, or consisting of a hexapeptide sequence having the sequence: P4-P3-P2-P1-X1-X2 where P4 is I or V; P3 is E, Q or M; P2 is any amino acid; P1 is D, X1 is A, S, W or R; and X2 is G, L or R.

[0006] The peptide is cleaved between the P1 and X1 amino acid residues. Throughout this disclosure, the conventional one-letter amino acid code is used to define amino acids.

[0007] Optionally, the hexapeptide sequence comprises, consists essentially of, or consists of the sequence: IEP / FD-X1-X2 where X1 is A, S, W, or R; and X2 is G, L, or R.

[0008] For the avoidance of doubt, P / F is understood to mean P or F.

[0009] The methods described herein can be used in a qualitative or quantitative sense. Thus, in certain embodiments, the methods can be used to measure Granzyme B activity.

[0010] After cleavage of the peptide, a cleaved peptide containing a detectable moiety is generated, which can be detected. A variety of detectable moieties can be envisioned, including radiometric, paramagnetic contrast agents, paramagnetic or superparamagnetic particles, and optically detectable moieties. In one conjugation, the detectable moiety is initially quenched when linked or bound to the peptide. However, following cleavage of the peptide by granzyme B, dequenching of the initially quenched signal occurs, and an increase in the detectable signal and a change in physicochemical properties is observed.

[0011] According to the present disclosure, it is possible to distinguish cleaved peptides from uncleaved peptides. One envisioned method employs the use of quenching moieties that are designed to quench a signal in the intact peptide, but are not quenched after peptide cleavage. It is also possible to use detectable signals that are not quenched or dequenched. In one embodiment, the uncleaved peptide is taken up by the cell, and since cleavage is performed internally, target cells with granzyme B show an increase in signal (e.g., fluorescence) and / or a change in physicochemical properties. In another embodiment, it is envisioned that the cleaved peptide may be taken up by the cell, but the uncleaved peptide is not internalized. In this way, the cleaved peptide can be detected following cellular uptake. The uncleaved peptide can have a partial or overall negative charge that prevents or reduces internalization by the cell. However, after the peptide is cleaved, the cleaved peptide does not contain that portion or negative charge, and therefore it may be internalized, allowing the cleaved peptide to be detected.

[0012] The probes of the present disclosure can be highly specific for Granzyme B. In this regard, highly specific means that the probe is more specific for Granzyme B than for other enzymes, such as other caspases, including caspase-3 and Granzyme A. Typically, the probes of the present disclosure have a K for Granzyme B of less than 30 μM, 25 μM, 20 μM, 15 μM, or 10 μM. M and / or 1×10 4 , 1×10 5 , 1×10 6 , or 1 × 10 7 Larger k cat / K M Indicates the value.

[0013] In one teaching, the detected signal may be an optical signal. The optical signal may be detected by a variety of methods known in the art, including visual and spectrophotometric methods, as well as the use of optical sensors, including CCD, CMOS sensors, photodiodes, and the like. Any suitable optical detection method may be employed and is not limited thereto.

[0014] According to one teaching of the present disclosure, the signal moiety may be a fluorescent moiety that is first quenched by the use of a quencher or a method of fluorescence resonance energy transfer (FRET). Detection of the fluorescent signal may be, for example, an increase in fluorescence intensity or a change in fluorescence lifetime. Fluorescence may be detected, for example, by fluorescence spectroscopy, fluorescence microscopy, confocal fluorescence microscopy, fluorescence image analysis, flow cytometry, laser scanning cytometry, a plate multiwell fluorescence reader, or a scintillation counter.

[0015] The principle of FRET, also known as Forster resonance energy transfer (FRET), resonance energy transfer (RET), and electronic energy transfer (EET), is based on the transfer of energy from an excited donor dye to a spatially closely located acceptor dye or quencher. A dark acceptor or dark quencher is a substance that absorbs the excitation energy from a fluorophore and dissipates the energy as heat; on the other hand, a fluorescent acceptor or fluorescent quencher re-emits much of this energy as light. Depending on the fluorescence quantum efficiency of the acceptor species, the energy transferred from the donor species to the acceptor species can either undergo non-radiative relaxation by internal conversion leading to quenching of the donor energy or be released by the fluorescence of the acceptor species.

[0016] FRET occurs between the electronic excited states of the donor and acceptor species when they are in sufficient proximity such that the excited state energy of the donor species is transferred to the acceptor species. As a result, the fluorescence period of the donor species is shortened and the fluorescence of the donor species is quenched. In one application of this principle, as applicable herein, the fluorescent moiety is placed in close proximity to the quenching moiety. In this arrangement, the energy from the excited donor fluorescent moiety is transferred to the acceptor quenching moiety and dissipated as heat rather than fluorescence. However, when the fluorescent moiety is not in close proximity to the quenching moiety, such as after cleavage of the peptide by granzyme B, the fluorescence of the fluorescent moiety is no longer quenched by the quenching moiety and can be detected, for example, by an increase in fluorescence intensity.

[0017] Thus, in accordance with one teaching of the present disclosure, a highly specific probe is provided for use in detecting granzyme B. The probe comprises a fluorescent moiety linked or attached to the peptide, and a quenching moiety linked or attached to the peptide, the peptide comprising, consisting essentially of, or consisting of a granzyme B cleavable hexapeptide sequence having the sequence: P4-P3-P2-P1-X1-X2 where P4 is I or V; P3 is E, Q or M; P2 is any amino acid; P1 is D or any IEP / FD X1 X2 where X1 is A, S, W, or R; and X2 is G, L, or R; Here, following cleavage of the peptide by Granzyme B, dequenching of the fluorescent moiety occurs and a detectable change in the fluorescent signal can be observed.

[0018] Since the present disclosure is directed to the cleavage of peptides by Granzyme B, the probes and methods described herein involve the detection of enzymatically active Granzyme B. Thus, the present disclosure can distinguish other teachings in which Granzyme B may not be active. For example, substrate binding without enzymatic cleavage does not indicate whether the binding molecule is enzymatically active. Furthermore, methods using antibodies that bind Granzyme B do not necessarily distinguish between activated and inactivated forms of Granzyme B. Thus, advantageously, the present invention can identify activated Granzyme B rather than the mere presence of Granzyme B, which may include inactivated forms. Furthermore, the present method can be used in combination with other methods to detect total Granzyme B levels. In this way, the ratio of activated Granzyme B to total Granzyme B can be calculated, and the amount of inactive enzyme can also be identified.

[0019] A detectable moiety, such as a fluorescent moiety, may be linked to a peptide by any suitable method. Typically, the detectable moiety is linked to the peptide by a covalent bond. For example, the detectable moiety may be linked to the peptide via an amide bond. The detectable moiety may be linked anywhere along the peptide sequence, but in one embodiment, the detectable moiety may be covalently attached to the N- or C-terminal amino acid of the peptide via an amide bond. In one embodiment, the signal-generating moiety may be covalently attached to the N-terminal amino acid. Linkage or covalent attachment via an amino acid side chain is also contemplated. The signal-generating moiety may be directly linked or covalently attached to the peptide, or may be attached via a linker molecule between the detectable moiety and the peptide.

[0020] When used, quencher moiety may be linked or bound to peptide in the same manner as detectable moiety, as described above.However, quencher moiety is generally not linked or bound to the same position on peptide as detectable moiety.In one embodiment, detectable moiety is linked or bound to the amino acid at N- or C-terminus, and quencher moiety is linked or bound to the amino acid at C- or N-terminus, respectively.

[0021] The detectable moiety, fluorescent moiety and / or quencher moiety may be indirectly linked to the peptide. For example, in one teaching, the fluorescent moiety and / or quencher moiety may be linked, bound and / or embedded within a nanoparticle, such as a latex nanoparticle, which may be bound to the peptide.

[0022] If used, suitable linker molecules include aliphatic compounds such as alkyl, alkenyl, or polyether chains, optionally C2-C 24 It has repeating units. The aliphatic molecule may, for example, contain one or more carboxylic acids and / or amino groups. Suitable aliphatic linkers include aliphatic diamines, such as 1,2-diaminoethane to 1,10-diaminodecane, and PEGylated diamines, such as 2,2'-oxydiethanamine to 1,8-diamino-3,6-dioxaoctane.

[0023] Alternatively, one or more naturally occurring or non-naturally occurring amino acids and / or imino acids can be used as linking groups.

[0024] Fluorescent moieties suitable for use in the present invention may include single molecules or molecular dyes. Dyes useful in the present invention include fluorescent hydrophobic dyes that fluoresce in the range of 400 to 1000 nm. Dyes include, but are not limited to, oxonols, pyryliums, squaric, croconic, rhodizone, polyazaindacene or coumarin, scintillation dyes (usually oxazoles, benzothiadiazoles, oxadiazoles), aryl- and heteroaryl-substituted polyolefins (C2-C8 olefin moieties), merocyanines, rhodamines, sulfocyanines, carbocyanines, phthalocyanines, oxazines, carbostyrils, porphyrin dyes, dipyrromethenaboron difluoride dyes, aza-dipyrromethenaboron difluoride dyes, and oxazine dyes. Fluorogenic dyes available commercially can be obtained from, for example, Thermofisher, Sigma Aldrich.

[0025] Exemplary quencher dye compounds suitable for use in the present disclosure can include single molecule or molecular dyes.Suitable quencher dyes include, for example, DABCYL and QSY series from Molecular Probes (www.probes.com), Dark Cy-dyes from Amersham Biosciences (www.amershambiosciences.com), Eclipse Dark Quencher dyes from Epoch Biosciences (www.epochbio.com), Black Hole Quencher dyes from Biosearch Technologies (www.biosearchtech.com), DYQ-dyes (www.dyomics.com), Black Berry Quencher from Berry&Associates (www.berryassoc.com), QXL Quencher from AnaSpec, Inc. and ElleQuencher from Oswel (www.oswel.com). Other quenchers include methyl red, Iowa Black FQ, Iowa Black RQ (Integrated DNA Technologies), IRDye QC-1 (Licor) and Si-rhodamine-based NIR dark quenchers (Mycohin et al., J. Am. Chem. Soc. 2015, 137, 14, 4759-4765).

[0026] The fluorescent moiety and quencher dye must be in sufficient proximity when attached to the peptide to ensure adequate quenching of any fluorescent signal prior to peptide cleavage. As shown here, sufficient quenching is observed when a hexapeptide is used and the fluorescent moiety and quencher moiety are attached to both ends of the peptide. One skilled in the art can easily test the quenching ability of longer peptides, as well as whether the fluorescent moiety and / or quencher moiety can be attached to the peptide termini or whether the fluorescent moiety and / or quencher moiety must be linked or attached via internal groups of the peptide.

[0027] In one embodiment, the peptide sequence comprises or consists of the following sequence: IEPD X1 X2, where X1 and X2 are defined as above.

[0028] In one embodiment, X1 is A, S, or W and / or X2 is G or L.

[0029] In one embodiment, the peptide is selected from the group consisting of: IEPDAG; IEPDSG; IEPDSL; IEPDWL; IEPDWG; IEPDAL; IEFDA L.

[0030] Probes of the present disclosure are capable of detecting Granzyme B in amounts of less than 10 nM, preferably less than 1 nM, less than 500 pM, less than 250 pM, less than 100 pM, or less than 50 pM.

[0031] It is desirable that the probe of the present disclosure exhibits rapid reactivity. In this context, rapid reactivity may refer to the ability to use granzyme B to cleave peptides in a short time. In one embodiment, this is the ability to cleave the percentage of peptides in a few hours, such as within 2 hours. In one test described herein, the test employed was to determine the percentage of peptides cleaved in 2 hours. In some embodiments, peptides that show 70% or more cleavage according to this test are desirable, and peptides that show at least 75% or 80% cleavage are desirable.

[0032] The probes as described herein may be provided in solution or may be bound to a substrate such as the wells of a microtiter plate, the surface of a microfluidic channel, a surface in a lateral flow system, or other suitable surface used in known analyte detection assays. The probes may also be contacted with granzyme B that is free in bodily fluids or excretions, cell samples, or biopsies, as well as granzyme B that has been first captured by the use of a granzyme B-specific binding agent as described herein.

[0033] Granzyme B-mediated cytotoxicity is the primary mechanism by which cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells eliminate pathogen-infected cells (including viral pathogens such as COVID-19), transformed cancer cells, epithelial cells such as those associated with IBD, and non-self cells in transplant rejection events such as kidney, liver or lung transplants.

[0034] Thus, the probes of the present disclosure can be applied in detecting immune-mediated cell death, such as cytotoxic activity of T and / or NK cells, in abnormal or unwanted cells. The abnormal or unwanted cells can be abnormally proliferating cells, such as malignant (i.e., cancer) or non-malignant tumor cells, and granzyme B is found in CD4+ T cells, mast cells, activated macrophages, neutrophils, basophils, dendritic cells, regulatory T cells, smooth muscle cells, chondrocytes, keratinocytes, type II pneumocytes, Sertoli cells, primary spermatocytes, granulosa cells, syncytiotrophoblast cells, as well as intestinal epithelial cells, virus- or bacteria-infected cells.

[0035] Granzyme B activity can also be detected extracellularly. GzmB is present in the serum of healthy individuals (approximately 20-40 pg / mL) and is increased, for example, in the serum of patients with HIV, Epstein Barr virus, arthritis and inflammatory bowel disease (IBD). GzmB is also found in the synovial fluid of patients with rheumatoid arthritis, the cerebrospinal fluid of patients with multiple sclerosis and in patients with Rasmussen's encephalitis. It is also found, for example, in the bronchoalveolar lavage of patients with COPD and those suffering from pulmonary inflammation or pulmonary sarcoidosis. GzmB is also associated with acute transplant rejection.

[0036] Thus, in one teaching, the use of a probe as described herein in detecting the level, e.g., abnormal level, of granzyme B in cells in a sample and / or body fluid as described above is provided. A suitable sample can include a tissue or a biopsy sample. The body fluid can include any suitable body fluid cerebrospinal fluid (CSF), whole blood, serum, plasma, cytoplasmic fluid, urine, feces, gastric fluid, digestive fluid, saliva, nasal or other airway fluid, vaginal fluid or semen, and most typically the biological body fluid is blood, plasma or serum. Typically, the sample is obtained from any suitable animal, most typically a mammal such as a human. In one embodiment, the disease / pathology to be detected is IBD, and the sample is a stool, or blood (e.g., serum or plasma) sample. In one embodiment, the pathology is acute transplant rejection, and the sample is a urine (for kidney), blood (serum or plasma), or lavage (for lung) sample. It is understood that abnormal levels can be identified by comparing to normal or baseline levels of Granzyme B obtained from non-disease cells or bodily fluids from subjects not suffering from the aforementioned identified diseases or conditions to determine whether the level of Granzyme B is substantially increased or decreased (or the same) relative to the normal or baseline level. It is understood that an increased or decreased level is at least 10%, 20%, 30%, 50%, 100%, 200% or more different from the normal or baseline level.

[0037] Thus, in one embodiment, stool sample is processed to provide a supernatant containing a portion of stool sample.The supernatant is contacted with a suitable granzyme B binding agent, such as anti-granzyme B capture antibody, which can be attached or adhered to a surface, so as to bind any granzyme B present in the portion of stool sample.Then, as defined herein, a peptide is contacted to detect and optionally quantify any granzyme B present in the portion of stool sample that is bound to anti-granzyme B antibody.

[0038] Other binding agents include antibody fragments capable of specifically binding to granzyme B, nanobodies, aptamers, and receptors or other proteins capable of specifically binding to granzyme B.

[0039] The surface may be any suitable surface, such as the surface of a slide, a microplate, the wall of a fluidic device, etc., but may also be, for example, the surface of a bead or a micro- or nanoparticle to which the binding agent is adhered, attached or otherwise immobilized.

[0040] The cells may be a single cell population or a diverse cell population, for example, as obtained from a tissue biopsy or specimen.

[0041] In further teachings, a method for detecting granzyme B in a cell or body fluid is provided, the method comprising contacting a probe described herein with a cell or body fluid sample isolated from a subject, in situ or in vitro, and detecting the level of granzyme B by cleavage of the peptide and release of a detectable signal moiety.

[0042] The probes of the invention may further be used in methods for detecting any effect (such as a therapeutic or cytotoxic effect) of an agent on a cell population, in vitro, ex vivo or in vivo.

[0043] [Detailed Description] The present disclosure will now be further illustrated, by way of example, with reference to the figures shown below. [Brief description of the drawings]

[0044] [Figure 1] Figure 1. Hexapeptide H5 achieves high reactivity and selectivity for GzmB by accessing a unique binding pocket. a) Fluorescence quantum yields of fluorescent hexapeptide H5 and intact and cleaved probes. b) Tetra- and hexapeptide sequences, fluorescence increase and t50 values ​​upon incubation with hGzmB. Data as mean ± SEM (n=3). c) Fluorescence over time (25 μM, black, 510 nm) of H5 (25 μM, dark grey, 510 mm) and Ac-IEPD-AMC (25 μM, light grey, 450 nm) after incubation with hGzmB (20 nM) at 37 °C. Data as mean ± SEM (n=5). d) Fluorescence change of peptide H5 (25 μM) after incubation with protease (20 nM) for 60 min at 37 °C. Data as mean ± SEM (n=3). e) Cleavage rate of peptide H5 by hGzmB (20 nM) as a function of substrate concentration (n=3). f) Representative binding modes of IEPD-Dabcyl at T1 (left) and IEPD-Dabcyl at H5 (right) based on MD simulations. g) Detailed interactions at the P1' and P2' sites of peptide H5 and overlay structures based on three independent experiments.

[0045] [Diagram 2]Figure 2. Probe H5 detects GzmB-mediated anticancer activity of CD8+ T cells. a) Schematic procedure of the co-culture assay. b) Representative microscopic images of CD8+ T cells before and after reactivation and staining with anti-GzmB (white arrows indicate fluorescent signals) and Hoechst33342 (nuclear outlines in grey) (n=3). Scale bar: 10 μm. c) Flow cytometry of E0771 cells in co-culture with activated (IL-2, 24 h) or inactive (IL-2, 2 h) CD8+ T cells or in monoculture in the presence of staurosporine (1 μm, 1 h): viable (white), stained with H5 (grey), apoptotic (black). (n=3). d) Confocal microscopy images of mKate-expressing E0771 cancer cells (nuclei show large round signals) stained with H5() in co-culture with activated T cells (left), non-activated T cells (middle) or activated T cells + Ac-IEPD-CHO (right). Black arrows highlight T cells and white arrows highlight intracellular GzmB puncta stained with H5. Scale bar: 10 μm. e) Time-lapse fluorescence microscopy images of OT-I CD8+ T cells (highlighted with letter T) killing OVA-EL4 cancer cells in the presence of H5 (becomes visible after t=2 h). Scale bar: 7 μm. f) Flow cytometry analysis of OVA-EL4 cancer cells from experiment e (n=3). P values ​​by two-tailed t-test.

[0046] [Diagram 3]Figure 3. Probe H5 detects T cell-mediated tumor regression in a squamous cell carcinoma mouse model. a) Experimental timeline of the CD8+ T cell-mediated tumor regression model. b) Cell populations found in wild-type SCC and SCC FAK(- / -) tumors. cf) SCC and SCC FAK(- / -) cells were injected into FVB immune-competent mice (1x106 cells / mouse) and tumors were harvested on day 14. Flow cytometry of wild-type SCC and SCC FAK(- / -) tumors for percentage of live cells by live / dead staining (c); CD8+ T cell infiltration by anti-CD8-PE (d); percentage of GzmB-positive SCC cancer cells by H5 staining (e); fluorescence intensity of H5 (525 nm) in SCC cancer cells (f). (n=4). SCC FAK(- / -) tumors (g) and wild-type SCC tumors (h) (stained ex vivo with 5 μM compound H5) were analyzed by flow cytometry and presented as pseudocolored two-dimensional tSNE (t-distributed stochastic neighbor embedding) plots to determine the distribution of the probe in the different cell populations (left) and the fluorescence intensity of H5 staining (right) (n=4). i) Representative images of SCC7.1 and SCC7.1 FAK - / - tumors harvested 14 days after cancer cell injection. j) Quantification of GzmB levels by ELISA in both tumors after tissue dispersion and protein extraction. GzmB levels were normalized to the total protein amount measured by the BCA method. Data are presented as mean ± SD (n=4). P values ​​were obtained from two-tailed t-test. j) Fluorescence emission in multiple cell subsets. Data are presented as mean ± SEM (two independent experiments).

[0047] [Figure 4]Figure 4. Probe H5 detects immunomodulatory effects in phenotypic screening and T cell-mediated cytotoxic activity in human lung cancer biopsies. a) Experimental protocol for phenotypic screening. b) Fluorescence intensity of probe H5 after co-culture of E0771 cells with IL-2-activated CD8+ T cells in the presence of small molecules (C1-C44). High concentrations of IL-2 were used as a positive control for activated CD8+ T cells, and rapamycin was used as a negative control. Probe H5 was incubated for 1 h and fluorescence images were obtained (n ≥ 8). Chemical structures of drugs showing H5 fluorescence signals equal to or greater than the positive control. c) Representative H&E microscopy images of healthy (left) and cancer (right) areas in biopsies taken from patients with lung adenocarcinoma. d) Cytometric analysis of EpCAM+H5+ cells in paired (healthy vs. cancer) biopsies from cancer patients after incubation with probe H5. (n = 3).

[0048] [Diagram 5] Figure 5. HPLC traces of probe H5 before and after reaction with hGzmB. HPLC chromatograms and mass spectrometry of probe H5 before and after reaction with increasing concentrations of hGzmB. Mcalc. (intact probe): 1246.6 [M+Na+]; Mcalc. (cleaved probe): 747.2 [M+H+].

[0049] [Figure 6] Figure 6. Comparison of enzyme selectivity and detection limit of hexapeptide H5. a) Fluorescence response of fluorescent peptides H5, T1 and Ac-IEPD-AMC after incubation with hGzmB (light grey bars), human caspase-3 (dark grey bars) and hGzmA (white bars) (n=3). b) Detection limit of hGzmB by fluorescence emission (530 nm) of hexapeptide H5 after reaction with stepwise increasing amounts of hGzmB (0, 9.6, 32, 96, 320, 960, 3,200, 9,600, 32,000 and 96,000 pg mL-1) at 37 °C. Data presented as mean ± SD (two independent experiments with at least three replicates each).

[0050] [Figure 7]Figure 7. Reactivity of hexapeptides H5 and H5m towards recombinant mouse GzmB. Fold increase in fluorescence of fluorescent peptides H5 and H5m (both at 25 μM) after 4 h preactivation with mouse cathepsin C followed by incubation with recombinant mouse pro-GzmB (100 nM) for 90 min at 37 °C. Excitation / emission wavelengths: 450 nm / 510 nm. Data presented as mean ± SEM (three independent experiments each with three separate replicates).

[0051] [Figure 8] Figure 8. Cell viability assay in mKate-E0771 cancer cells. mKate-E0771 cells were plated in 96-well plates (50,000 cells well-1) and incubated with the indicated concentrations of probe H5 for 1 h at 37°C. Cell viability was measured using a commercial MTT kit (Invitrogen) with values ​​normalized to untreated cells. Data are presented as mean ± SEM (two independent experiments with triplicates each).

[0052] [Figure 9] Figure 9. Co-culture of OT-I CD8+ T cells with OVA-EL4 cancer cells. a) Schematic of the immunological synapse between CD8+ T cells and cancer cells, highlighting the fluorescent staining of peptide H5 (green) before cells undergo apoptosis and the dead cell marker Sytox Blue (blue) after granzyme B initiates apoptosis. b) Longitudinal fluorescence emission of probe H5 (1 nM) upon acute increase of hGzmB at time 0 and at different time points (1 nM increase of hGzmB) highlighted by the arrows (n=3).

[0053] [Figure 10]Figure 10. Probe H5 detects CD8+ T cell-mediated cell death of cancer cells induced by IL-2 and AZD5363. a) Representative histograms of probe H5 staining in E0771 cells alone (50,000 cells well-1) or in co-culture with CD8+ T cells (200,000 cells well-1) and treated with AZD5363 (1 μM). Excitation / emission wavelengths: 488 nm / 525 nm. b) Representative flow cytometry contour plots () of E0771 cells (50,000 cells well-1) after co-culture with mouse CD8+ T cells (200,000 cells well-1) and dual treatment with IL-2 (200 U mL-1) and AZD5363 (1 μM). Co-cultured cells were stained with probe H5 (5 μM) and AF647-Annexin V (10 nM). Excitation / emission wavelengths: 488 nm / 525 nm (probe H5), 633 / 670 nm (AF647-Annexin V). c) Percentage of E0771 cells double stained with probe H5 and AF647-Annexin V under the experimental conditions described in a). Data are presented as mean ± SEM (n=3).

[0054] [Figure 11] Figure 11. A) Fluorescence response (n=3) of probes H5 or R7 (25 μM, filled circles, filled squares) after 1.5 h incubation in urine with recombinant human granzyme B (15 nM) or H5 or R7 (H5: triangles, R7: inverted triangles) incubated without granzyme B in urine. HPLC chromatograms showing R7 (panel B) and H5 (panel C) after 3 h incubation in human urine with (bottom) or without (top) granzyme B at 37 °C. D) Detection limit of human recombinant granzyme B spiked into human urine after incubation with probe H5 at 37 °C for 3 h. E) Detection limit of human recombinant granzyme B spiked into human urine after incubation with probe R7 at 37 °C for 3 h.

[0055] [Figure 12] Figure 12. Schematic diagram of the in-house developed Granzyme B antibody capture assay. Excitation / emission H5: 450 / 510 nm, R7: 620 / 660 nm.

[0056] [Figure 13] Figure 13. Mean concentration of Granzyme B per group measured by interpolation of the Granzyme B standard curve after 18 hours of incubation at 37°C using the method outlined in Figure 12 with probes H5 (A) or R7 (B) (both at 25μM) and healthy stool (n=46) and IBD patient (n=48) stool samples. H5λexc / emi: 450 / 510nm. R7λexc / emi: 620 / 660nm. Significant differences, p-values ​​calculated by unpaired t-test, are shown on the graph. Calprotectin levels above 500ug / mg samples are classified as high calprotectin. Calprotectin levels below 100ug / mg are classified as low calprotectin.

[0057] [Figure 14] Figure 14. A) Fluorescence fold change of probe R1-10 (25 μM) incubated with human recombinant granzyme B (20 nM) for 2 h at 37 °C (n=6). Error bars are SEM. B) Representative HPLC spectra of probe R7 before (top) and after (bottom) incubation with granzyme B for 2 h at 37 °C. C) Fluorescence fold change of probe R7 after incubation with selected enzymes (Casp-3: caspase-3, GzmA: granzyme A, GzmB = granzyme B) including granzyme B previously inhibited with a commercial inhibitor (Ac-IEPD-CHO, Enzo life sciences) (n=3). Error bars are SEM. D) Fluorescence kinetics and associated kcat / Km values ​​of R7 (25 μM) after incubation in the presence (grey) or absence (black) of granzyme B (n=3). Probe excitation: 620 nm, probe emission: 660 nm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] [method] [Chemical synthesis] C-terminal functionalization procedure To a solution of peptide fluorophore conjugate (1 eq) in CHCl:DMF (1:1, 1 mL) was added benzyl (2-aminoethyl)carbamate (2 eq), Oxyma (2.5 eq) and PyOxim (2.5 eq). The reaction mixture was stirred at -20°C for 5 min. DIPEA (5 eq) was then added and stirring was maintained at -20°C for 3 h. The reaction was allowed to warm to room temperature. The solvent was removed under reduced pressure. The residue was purified by semi-preparative HPLC to give the purified peptide.

[0059] General procedure for hydrogenation Fluorescent peptide (1 eq) and Pd / C (10%) (0.5 eq) or Pd(OH)2 / C (20%) (0.5 eq) were dissolved in 2% formic acid in MeOH (5 mL) that had been previously purged with N2. The reaction vessel was flushed with N2, evacuated, and filled with H2 gas. The reaction mixture was stirred under H2 gas at room temperature. Stirring was continued for 2 h at atmospheric pressure. The reaction mixture was then filtered through Celite to remove the catalyst, and the filtrate was evaporated under reduced pressure to isolate the deprotected peptide.

[0060] General procedure for Dabsyl coupling To a solution of Dabsyl-OSu (1.2 eq) in CHCl:DMF (1:1, 1 mL) was added peptide (1 eq) and DIPEA (2 eq). Stirring was maintained at room temperature for 24 h. The solvent was removed under reduced pressure and the residue was dissolved in MeOH and purified by semi-preparative HPLC to give the final peptide.

[0061] Benzyl (2-aminoethyl)carbamate [ka] Benzyl chloroformate (428 μL, 3 mmol) in CHCl (10 mL) was added dropwise to a solution of 1,2-diaminoethane (2 mL, 30 mmol) in CHCl (40 mL) at 0° C. over 1 h. The reaction was stirred at 0° C. for 1.5 h and then at room temperature overnight. TLC analysis (CHCl:MeOH, 7:3) showed the reaction was complete. The precipitate formed in the reaction was removed by filtration, and the filtrate was then washed with brine, dried over MgSO4, and the solvent was removed under reduced pressure to give the compound as an amorphous yellow solid (570 mg, 98% yield). The crude product was used in the next step without further purification. 1 H NMR (500MHz, CD3OD) δ7.44-7.25(m,5H), 5.09(s,2H), 3.22(t,J=6.2Hz,2H), 2.75(t,J=6.2Hz,2H). 13 C NMR (126MHz, CD3OD) δ157.7,136.9,128.0,127.5,127.4,66.0,42.6,40.8. MS (ESI+, HO / MeCN): [M+H + ]calcd.C 10 H 15 For N2O2: 195.1; found: 195.3. All spectral characteristics are from the literature.

[0062] 2,5-Dioxopyrrolidin-1-yl 4-((4-(dimethylamino)phenyl)diazenyl)benzoic acid (Dabcyl-OSU) [ka] N-Hydroxysuccinimide (85 mg, 0.74 mmol), 4-((4-(dimethylamino)phenyl)diazenyl)-benzoic acid (100 mg, 0.37 mmol) and EDC HCl (142 mg, 0.74 mmol) were dissolved in a mixture of CHCl:DMF (1:1, 10 mL) and stirred at room temperature for 16 h. The solvent was then removed under reduced pressure to give the crude product. The crude product was purified by flash column chromatography (CHCl:MeOH, 99:1) to give the compound as an orange solid (100 mg, 88% yield). 1 H NMR(500 MHz,DMSO-d6)δ8.25-8.20(d,J=8.6Hz,2H),7.98-7.93(d,J=8.7Hz,2H),7.89 -7.84(d,J=8.7Hz,2H),6.90-6.85(d,J=8.8Hz2H),3.11(s,6H),2.91(s,4H). 13C NMR (126MHz, DMSO-d6) δ170.8,161.9,157.0,153.9,143.2,131.9,126.2,124.4,122.9,112.1,26.0. MS(ESI+,HO / MeCN):[M+H + ]calcd for C 19 H 19 N4O4:366.1;found:366.0.

[0063] Probe H5 [ka] 1H NMR(500MHz,MeOD)δ7.98(d,J=8.5Hz,2H),7.86(dd,J=10.2,8.8Hz,4H),7.42(s,1H),7.01(d,J=4.1Hz,1H),6.86(d,J=9.3Hz,2H),6.33(d,J=4.0Hz,1H),6.22(s,1H),4.68(dd,J=8.7,5.4Hz,1H),4.55(t,J=6.4Hz,1H),4.39-4.29(m,2H),4.25-4.13(m,2H),3.93-3.83(m,1H),3.81-3.74(m,1H),3.65-3.53(m,3H),3.47(t,J=5.9Hz,2H),3.24(t,J=7.6Hz,2H),3.01(s,1H),2.88(s,1H),2.83(d,J=6.4Hz,2H),2.77-2.65(m,3H),2.52(s,3H),2.46(t,J=7.1Hz,2H),2.29(s,3H),2.22-2.15(m,2H),2.11-1.99(m,2H),1.98-1.90(m,3H),1.85-1.62(m,5H),1.52-1.47(m,2H),1.45(d,J=7.3Hz,3H),1.38(d,J=7.2Hz,1H),1.35-1.30(m,3H),1.15(m,2H),1.00-0.74(m,12H). 13 C NMR(126MHz,MeOD)δ174.1,173.9,173.2,172.5,168.4,157.0,155.0,153.2,143.4,134.3,133.5,128.2,128.1,125.0,124.3,121.5,119.9,116.4,111.2,61.2,57.8,57.6,52.3,50.9,50.7,39.7,39.2,39.0,38.7,38.636.5,34.2,29.3,29.0,28.9,24.8,24.7,24.6,24.1,23.9,22.1,20.1,15.9,14.5,13.5,9.9,9.7. HRMS:[M+Na + ]calcd. for C 60 H 80 BF2N 13 NaO 12 :1246.5989;found:1246.6013.

[0064] Calculation method A simulation system was constructed from the crystal structure of GzmB with PDB id 1IAU. Probes T1 and H5 were constructed using Maestro, and the peptide backbone of the IEPD moiety was superimposed onto that of the co-crystallized inhibitor (Ac-IEPD-CHO) with side chains and Dabcyl manually adjusted to avoid steric clashes. The atom types of the peptide fragments of the protein and probe were assigned using the FF14SB force field. The linker and Dabcyl quencher were parameterized by GAFF2 atom types. Three disulfide bonds were constructed between Cys pairs 49-65, 142-209 and 173-208. Each system consisting of protein and probe was solvated in a truncated octahedral TIP3P water box with a buffer region of 12 Å. Necessary counterions were added to neutralize the system, and a minimization step was performed using 3,500 iterations of steepest descent with 6,500 iterations of the conjugate gradient algorithm. Prior to production, three independent replicates were prepared for each system (i.e., Gmzb-T1 and GzmB-H5) and each replicate was heated in three steps for 150 ps (50 K to 150 K, 150 K to 250 K, 250 K to 298 K) in the standard ensemble with a time step of 1 fs. The density was subsequently equilibrated for 500 ps in the NPT ensemble with a time step of 2 fs. The Langevin thermostat (collision frequency 3 ps -1 ) and a Montecarlo barostat were used to maintain temperature and pressure. Throughout the heating and equilibration steps, the distance between Ser283 and the carbonyl of the Asp residue in the probe was kept within the flat-bottom restraint (k = 5 kcal mol -1 Å -2 ) was used to keep the spacing below 4.0 Å. Production runs for each replicate consisted of 200 ns long simulations in the NPT ensemble using a 2 fs time step.

[0065] Fluorescence assays with recombinant enzymes. Fluorescence assays with enzymes were performed in buffer-1 (50 mM Tris, 100 mM NaCl, pH 7.4) for GzmB, pro-GzmB, GzmA and HNE, and in buffer-2 (25 mM HEPES, 0.1% CHAPS, 10 mM DTT, pH 7.5) for caspases and other enzymes. Probes (25 μM) were added to enzymes (20 nM or the indicated concentrations) in 384-well plates, and their fluorescence emissions were recorded at 450 nm (for AMC) or 510 nm (for BODIPY) at 37 °C using a SynergyH1 hybrid reader (BioTek). In experiments with Ac-IEPD-CHO (50 μM), the inhibitor was preincubated with GzmB for 1 h before adding the probe.

[0066] Primary cell isolation and cell culture. E0771 cancer cells expressing the nuclear-localized red fluorescent protein mKate were cultured in 10% fetal bovine serum (FBS), antibiotics (100 U mL -1 Penicillin and 100mgmL -1 The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 0.1 mM EDTA (1 mM streptomycin) and 2 mM L-glutamine in a humidified atmosphere at 37°C with 5% CO2. Cells were routinely passaged into T-25 cell culture flasks upon reaching 90% confluency. Primary CD8+ T cells were isolated from mouse spleens by tissue homogenization, red blood cell lysis, and purification with magnetic beads (CD8 MicroBead Kit).

[0067] Computational studies and molecular dynamics simulations. The model was built with Maestro and AMBER16 using PDB id 1lAU. The force fields ff14SB, GAFF2 and TIP3P were used to parameterize the solvated system. Torsional parameters for the azobenzene moiety in Dabcyl were provided by the Luque research group. 5 For each system, a Langevin thermostat (collision frequency 3 ps -1) at 298 K and 1 atm for 200 ns, with three iterations performed, and the Monte Carlo barostat was implemented in PMEMD. 6 .

[0068] Flow cytometry. E0771 (5 × 10 4 cells / well) with IL-2 (1,000 U mL -1 ) on a Geltrex-coated 6-well plate and pre-injected with anti-mouse CD3e (2 μg mL -1 ), anti-mouse CD28 (5 μL mL -1 ) and IL-2 (80 U mL -1 Mouse CD8 T cells (2.5 × 10 5 Cells were co-cultured with either 0.1% glycerol (0.1% glycerol / well) or 0.1% glycerol (0.1% glycerol / well). Treatment with staurosporine (1 μM) was performed for 1 h. Flow cytometry preparation included trypsin-EDTA (0.05%) treatment, washing, resuspension in PBS and incubation with probe H5 (5 μM) for 1 h at 37 °C. Cells were then washed twice and incubated with Annexin V-AF647 (10 nM) before flow cytometry analysis in a 5L LSR with data analyzed in FlowJo. Excitation / emission wavelengths: H5 (488 nm, 525 ± 50 nm), mKate (561 nm, 635 ± 15 nm), Annexin V-AF647 (640 nm, 670 ± 14 nm).

[0069] Live cell fluorescence confocal microscopy. IL-2 (80U mL -1 ), anti-mouse CD3e (2 μg mL -1 ) and anti-mouse CD28 (5 μL mL -1 ) in concentrated E-DMEM phenol red-free medium containing E0771 (1.5 × 10 3 cells / well) with freshly isolated murine CD8+ T cells (1.2 × 10 4 After 48 h, IL-2 (1,000 U mL -1T cells were reactivated by addition of Ac-IEPD-CHO at 25 μM and incubation for 3 h. Probes H5 at 25 μM and Ac-IEPD-CHO at 50 μM were used with 1 h preincubation. After 1 h of treatment with H5 at 37 °C, cells were washed with medium and imaged under a Leica TCS SP8 fluorescence confocal microscope with a 40x oil immersion objective. Excitation wavelengths: 488 nm (H5), 561 nm (mKate). Images were analyzed with ImageJ.

[0070] Preclinical cancer models. SCC tumors were established and stained by flow cytometry as previously described. 7 Simply put, 1×10 6 SCC cancer cells were injected subcutaneously into FVB / N mice, sacrificed 14 days after implantation, and tumors were dissociated to generate single cell suspensions. Cells were stained with antibodies and probe H5 for 30 min at room temperature and analyzed by flow cytometry (BD Fortessa). Data analysis was performed using FlowJo software.

[0071] Drug screening. E0771 (750 cells / well) and freshly isolated murine CD8+ T cells (6,000 cells / well) were plated in 384-well plates as described above. After co-culture at 37 °C for 2 days, IL-2 alone (100 or 250 U mL -1 ) or IL-2 (100 U mL -1 ) and drugs (Table 3) were added for another 3 h. H5 (20 μM) and Hoechst33342 (1 μM) were added for 1 h at 37 °C, and after washing with PBS, the cells were imaged using ImageXpress with KRB (10% FBS) buffer as the medium. TMImages were acquired under XLS (Molecular Devices). Fluorescent images (4 sites / well) were acquired under a 40x objective lens using 405 nm (Hoechst) and 488 nm (H5) excitation wavelengths, and data were analyzed in MetaXpress software with a custom module editor. Briefly, nuclei were used as seeds to create pseudocellular regions, and Granzyme B positive puncta were detected using a top-hat filter (pixel size: 10, circle shape) followed by an average filter (3 x 3 pixels).

[0072] Human biopsies. Tissue samples were taken from treatment-naïve patients undergoing surgical resection. Participants provided written informed consent and the study was approved by NHS Lothian REC and proceeded by the NHS Lothian SAHSC Bioresource (record number: 15 / ES / 0094). Fresh samples were minced and diluted with 1 mg / mL collagenase IV, 1 mg mL -1 DNase 1, 50 U mL -1 Red blood cell lysis was performed after 1 h at 37°C in DMEM containing 100 μm hyaluronidase and passage through 100 and 70 μm filters. For microscopic examination, formalin-fixed paraffin-embedded slides of non-small cell lung cancer and paired non-cancerous lungs were deparaffinized, rehydrated, stained with hematoxylin and eosin, subsequently dehydrated, and imaged on a Zeiss AxioScan microscope.

[0073] Fluorescence analysis in urine samples The probes (H5 or R7) (25 μM, 50 μM) in Granzyme B buffer (50 mM Tris, 100 mM NaCl, pH 7.0) were incubated with a range of Granzyme B concentrations (20,000 pg mL -1 ~78pg mL -1 Granzyme B was added directly to urine samples (50 μL) from healthy donors containing granzyme B or no granzyme B (control). The solutions were incubated at 37° C. and the fluorescence response was monitored over time (λ exc / emFluorescence was read on a Synergy Biotech H1 spectrophotometer.

[0074] HPLC / MS analysis of probe cleavage by urinary granzyme B After incubation of the probes (H5 or R7) (25 μM) with human recombinant granzyme B (15 nM) in granzyme B buffer for 1.5 h at 37 °C, the samples were diluted with MeOH, centrifuged, and the supernatants were injected into the HPLC / MS. Samples were eluted with 0.1% HCOOH in HO (A) and 0.1% HCOOH in MeCN (B), with a gradient from 0 to 100% B over 5 min, followed by an isocratic period of 3 min, at a flow rate of 1.5 mL min -1 The reaction was monitored from 220-700 nm, and the percentage cleaved was determined by comparing the peak area of ​​the cleaved probe to the intact probe on the MassLynx software.

[0075] Fluorescence analysis in stool samples High-binding microplates (Merck) were coated with 50 μL (2 μg / ml) of anti-GzmB antibody (human, QuickZyme Biosciences) in coating buffer (NaOAc buffer, pH 5.5) and incubated overnight at 4°C in a humidified chamber. The plates were washed four times with wash buffer (0.01M PBS, 0.05% (v / v) Tween 20, pH 7.5) and the antibodies were blocked by adding 100 μL of Chonblock to the 96-well plate for 1 h at room temperature. The plates were then washed with wash buffer (×4) and clinical samples diluted in Chonblock (1:1) were added to the plate (50 μL) along with Granzyme B standards also diluted in Chonblock (1:1). Samples were incubated for 2 h at room temperature before the plates were washed with wash buffer (×4). Finally, probes H5 or R7 (25 μM) were added to each well (total volume 50 μL) and the fluorescence intensity was monitored at 0, 2, 4, 18 and 24 h using a Synergy H1 Biotek microplate reader. H5:λex = 470 nm and λ ex = 510 nm, and a monochromator with a gain setting of 90 was used for R7:λ ex = 620 nm and λ ex = 660 nm. EXAMPLES

[0076] In this study, we rationally designed GzmB substrates and identified new hexapeptide sequences that optimally fit into the active site of the enzyme, suggesting alternative binding modes as highly specific GzmB probes. Optimization of the hexapeptides into FRET constructs was achieved by using the k-terminal sequence of the IEPD tetrapeptide. cat / K M This resulted in probe H5, which is several orders of magnitude better than the ELISA ratio. The high speed and selectivity of H5 enabled real-time measurement of anticancer activity of T cells in a mouse model of immune-mediated tumor regression. We also showed that H5 can be used in image-based screening to identify new drugs capable of reactivating CD8+ T cells against cancer cells. Finally, we optimized probe H5 in biopsies from cancer patients to detect in situ T cytotoxic activity in human tumors.

[0077] Design of highly reactive fluorescent probes for granzyme B The tetrapeptide IEPD is a key scaffold that has been reported for the preparation of covalent inhibitors as well as fluorescent substrates targeting human GzmB (hGzmB). 8-10 We first assessed the reactivity of commercially available Ac-IEPD-AMC (i.e., a substrate that releases 7-amino-4-methylcoumarin upon reaction with hGzmB) to enzyme concentrations applicable to clinical assays. We observed slow enzyme cleavage rates for Ac-IEPD-AMC (i.e., less than 1% cleavage after 2 h reaction with 20 nM enzyme, and 10% cleavage after 24 h reaction with 100 nM) and a limit of detection (LoD) of 25 nM, which is far from the pM concentrations of GzmB found in clinical samples. 11Given these results, we investigated whether IEPD-based Forster resonance energy transfer (FRET) probes would show increased reactivity towards hGzmB. FRET substrates were synthesized by flanking the IEPD sequence with a fluorophore and a quencher as a donor-acceptor pair. Unlike Ac-IEPD-AMC, the fluorophore was placed at the N-terminus and the quencher next to the cleavage site, so that its electron-withdrawing properties favored enzymatic cleavage. Different combinations of fluorophore, spacer and quencher were synthesized and the tetrapeptide T1 (with BODIPY-FL as the fluorophore and ethylenediamine-dabsyl as the quencher) was identified as the most reactive substrate. Nevertheless, the reactivity of T1 towards hGzmB was low (i.e., <5% cleavage in a 2-h reaction with 20 nM enzyme and 15% cleavage in a 24-h reaction with 100 nM enzyme) with a LoD of 17 nM.

[0078] We therefore decided to optimize the FRET substrates by identifying sequences that could react faster and more specifically with hGzmB. We therefore prepared FRET hexapeptides in which the IEPD sequence was extended with small amino acids at the P1' and P2' positions (Fig. 1a). Hexapeptides H1 (IEPDAG) and H2 (IEPDSG) showed significantly faster conversion than tetrapeptide T1 (e.g., 75% and 80% conversion, respectively, Table 2), but conversion was incomplete upon 2 h treatment with 20 nM hGzmB. As peptide H2 showed slightly higher reactivity, we prepared peptide H3 (IEPDSL), which retained the serine at the P1' position and had a more hydrophobic leucine at the P2' position. As this change did not improve reactivity (e.g., 80% conversion, Table 2), alternatives at the P1' and P2' positions were explored. Although hexapeptide H4 achieved 83% conversion, peptide H5 showed the highest reactivity, showing fast and complete cleavage in less than 30 min (Table 2 and Figure 5). Interestingly, the results suggested that smaller amino acids (i.e., alanine instead of tryptophan) were better tolerated by the enzyme. To confirm this observation, negative control peptides of the H4 and H5 sequences were synthesized, containing arginine at P1' or P2' to inhibit binding. The resulting hexapeptides H6 (IEPDWR) and H7 (IEPDRL) showed significantly lower reactivity (i.e., 40% and 60% conversion, respectively, Table 2), but much higher reactivity than tetrapeptide T1. These results suggest that the FRET hexapeptide construct reacts with hGzmB much faster than the shorter tetrapeptides. We prepared all peptides using solution and solid-phase synthesis (as described below), isolated them by preparative HPLC at >95% purity, and confirmed their identity by high-resolution mass spectrometry.

[0079] All peptide conjugates were synthesized using the Fmoc / tThe peptides were synthesized using solid-phase peptide synthesis using the Bu strategy. Resin loading was performed with the appropriate Fmoc-protected amino acid (1.4 eq) and DIPEA (10 eq) in DCM at room temperature for 1 h. The remaining 2-chlorotrityl groups were then capped with MeOH (0.8 μL / mg resin). Fmoc removal was performed with piperidine (20% in DMF) and Oxyma (1.0 M) to minimize aspartimide formation under 3 × 5 min cycles. Amino acids were coupled with an excess of Fmoc-protected amino acid (4 eq), COMU (4 eq), Oxyma (4 eq), and DIPEA (8 eq) in DMF at room temperature for 1.5 h. Completion of coupling was monitored by the Kaiser test (and chloranil test after proline coupling). The aspartic and glutamic acid side chains were protected with OBzl groups and the arginine side chain with NO2 groups, since they can be removed under mild Pd-catalyzed hydrogenation tolerated by the acid-labile BODIPY-FL fluorophore. Fluorophore attachment was performed on the solid phase with BODIPY-FL (1.1eq), COMU (1.15eq), Oxyma (1.15eq) and DIPEA (3eq) in DMF for 1.5 h at room temperature. Resin cleavage was performed by treating the resin with TFA (1% in DCM) for 5 × 1 min cycles. The combined solution was then poured onto DCM and evaporated under reduced pressure. Purification was performed by semi-preparative HPLC and the pure fluorescent peptide was coupled to Cbz-protected 1,2-diaminoethane. Catalytic hydrogenation to remove all protecting groups was then performed to allow the introduction of the quencher Dabsyl to the terminal amine of the linker via NHS-mediated coupling. The final peptide, with a purity of >95%, was isolated by semi-preparative HPLC.

[0080] [ka] Probe H5 is a highly specific substrate of human GzmB by accessing a unique binding pocket.

[0081] We analyzed the fluorogenic response of all hexapeptides H1–H7 by measuring their fluorescence emission in the presence of recombinant hGzmB (Fig. 1a). As expected, all hexapeptides outperformed tetrapeptide T1, with signal-to-background ratios ranging from 12-fold (peptide H6) to 153-fold (peptide H5) (Fig. 1b). We also found that the most reactive compound exhibited a 50-fold increase in fluorescence (t 50 The time required for the peptide to reach a t 50 The kinetic properties of the hexapeptide H5 were also compared with previously reported tetrapeptide constructs. Peptide H5 exhibited high μM min -1 Range of V max value and 1.2×10 7 M -1 s -1 k cat / K M The results showed a remarkably high catalytic efficiency at the 1000-fold ratio (Fig. 1e) and a more than 1000-fold improvement over the fluorescent tetrapeptide (Table 1). These results confirmed the exceptional reactivity of probe H5, showing an unprecedented LoD of 6 pM against hGzmB (Fig. 6b).

[0082] We also evaluated the specificity of peptide H5 to hGzmB against inactive pro-hGzmB and other enzymes, including other serine proteases (e.g., granzyme A, neutrophil elastase) and cysteine ​​proteases active during apoptosis (e.g., caspases). Probe H5 showed minimal response to other enzymes (Figure 1d), including caspase-3 (Figure 6), which can cleave some IEPD-based probes. This represents one important advantage when assessing the response to immunotherapy, since cross-reactivity with caspase-3 would prevent distinguishing between global cell death and T-cell-mediated cancer cell death. Finally, to further confirm that the fluorescence emanating from probe H5 was due to a specific reaction with hGzmB, we performed experiments in the presence of the irreversible GzmB inhibitor Ac-IEPD-CHO, which dramatically reduced the fluorescence response of H5 (Figure 1d).

[0083] To understand the difference in reactivity between the model tetrapeptide T1 and the novel hexapeptide, we used molecular dynamics (MD) simulations to compare the preferred binding modes of T1 and H5 in complex with the hGzmB model. Because all hexapeptides shared P1-P4 amino acids with BODIPY-FL, we focused on the analysis of the P1'-P2' residues and the Dabcyl quencher. MD simulations revealed that T1 and H5 accommodate the quencher in two different pockets. In the hGmzB-T1 simulation, the Dabcyl moiety preferentially bound in the groove between loops 74-84 and loops 146-161 (Fig. 1f), whereas in the hGmzB-H5 simulation, the quencher preferentially bound in the extension of the catalytic groove depicted by loops 74-84 and β-barrel 35-114 (Fig. 1f). Visual inspection of the trajectories revealed that the backbone of residues 200-201 forms a saddle point, highlighted in orange in Fig. 1f, that prevents the short tetrapeptide T1 from accommodating the quencher along the catalytic groove. In both cases, the Dabcyl moiety remains flexible and does not form long-lived interactions with specific protein residues. MD simulations also suggest that the higher reactivity of H5 compared to the other hexapeptides is due to the combined effect of the alanine residue at P1', which fills a small hydrophobic pocket in the catalytic groove, and the leucine residue at P2', which ties up the saddle point at 200-201 (Fig. 1g). This binding mode explains the enhanced reactivity of the H5 peptide to: 1) peptides H4 and H6, whose P1' tryptophan is too large to fit into the pocket; 2) peptide H7, whose P1' arginine is electrostatically disadvantaged by the basic residues that define this subpocket; 3) peptide H3, whose P1' serine lacks an adjacent effective hydrogen bond donor; and 4) peptides H1 and H2, whose P2' glycine is too small to pack effectively against the saddle point at 200–201.

[0084] Probe H5 detects real-time reactivation of T cells in co-culture with cancer cells Given the reactivity and selectivity of probe H5 for GzmB, we investigated its utility for measuring the cytotoxic activity of T cells attacking cancer cells. To investigate this, we co-cultured mouse CD8+ T cells with E0771 breast cancer cells (Figure 2a). Although the substrate for mouse GzmB features a phenylalanine at P2 instead of the proline of the human substrate, a good response of probe H5 to mouse GzmB was observed, as was the case with a hexapeptide analog containing a phenylalanine (probe H5). m:IEFDAL, Figure 7). Therefore, we decided to use probe H5 for both mouse and human assays. First, we optimized the co-culture to effectively increase the levels of GzmB in CD8+ T cells. Incubation with the interleukin IL-2 resulted in the greatest reactivation, and we confirmed by fluorescence microscopy with anti-GzmB that more than 80% of CD8+ T cells expressed inactive GzmB after IL-2 treatment (Figure 2b). Next, we applied these conditions to co-cultures of CD8+ T cells and genetically modified E0771-La2-NLR cells expressing the red fluorescent protein mKate to facilitate detection, and incubated with probe H5 and annexin V-AF647, a marker of apoptosis. Flow cytometry analysis revealed that more than 80% of cancer cells co-cultured with activated T cells were double stained with H5 and annexin V-AF647, confirming that the release of probe H5 indicates immune-mediated cancer cell death (Figure 2c). Cancer cells co-cultured with non-reactivated T cells or treated with staurosporine, a kinase inhibitor that induces apoptosis, showed weak fluorescence, confirming that probe H5 detects dead cancer cells killed by activated CD8+ T cells (Figure 2c). Fluorescence microscopy experiments also showed that probe H5 stained only the cytoplasm of mKate+ cancer cells, but not T cells (Figure 2d). As an additional control, probe H5 was not cytotoxic (Figure 8) and did not stain cancer cells in co-cultures with inactive T cells or co-cultures pretreated with the irreversible GzmB inhibitor Ac-IEPD-CHO (Figure 2d). Overall, these results confirm that probe H5 can detect cancer cell death resulting from the attack of activated CD8+ T cells and suggest that the fluorescence emission of probe H5 can be used as a biomarker of immunomodulatory effects in cultures under viable conditions. We next asked whether H5 could be used to image in real time how CD8+ T cells attack cancer cells. We utilized CD8+ T cells expressing the OT-I transgenic receptor, which specifically targets cells presenting the OVA-derived SIINFEKL antigen in the context of H-2kb, and co-cultured them with SIINFEKL-loaded EL4 cancer cells (Figure 9). Cells were counterstained with Cell Tracker Orange for easier identification and incubated with probe H5 and the cell death marker Sytox Blue, followed by time-lapse microscopy. Initially, probe H5 remained unchanged, but its release within the target cells gradually increased as CD8+ T cells began to form immune synapses with the cancer cells (Figures 2e and ). After several contacts between CD8+ T cells and cancer cells, the green fluorescent signal of H5 colocalized with the blue signal of Sytox Blue, indicating that the target cancer cells underwent GzmB-mediated apoptosis. Quantitative flow cytometry analysis also confirmed that the fluorescent signal of peptide H5 in cancer cells preceded that of Sytox Blue, supporting active GzmB as an early biomarker of immune-mediated cancer cell death (Figure 2f). In parallel, we performed in vitro experiments mimicking the intracellular environment found in cancer cells receiving multiple contacts from CD8+ T cells and observed the cumulative fluorescent signal of probe H5 for a 1 nM increase in spikes of GzmB (Figure 9).

[0085] Probe H5 detects T cell-mediated tumor regression in a mouse model of cancer. We further investigated the ability of peptide H5 to detect GzmB activity in a mouse model of tumor regression. Serrels et al. reported that inhibition of focal adhesion kinase (FAK) promotes T cell-mediated regression of squamous cell carcinoma (SCC) tumors through modulation of the immunosuppressive microenvironment (Figure 3a, b), and FAK inhibitors are currently in clinical trials in combination with immune checkpoint inhibitors. 12Because tumor regression in SCC FAK(- / -) mice is dependent on CD8 T+ cells, this preclinical model is an excellent platform to examine whether probe H5 can detect T cell-mediated cancer cell death in tumors.

[0086] First, FVB immune-competent mice were administered SCC FAK(- / -) cancer cells or wild-type SCC cancer cells (as a negative control) and tumors were allowed to grow for 2 weeks. As expected, SCC FAK(- / -) tumors were significantly smaller and contained less viable cells than wild-type SCC tumors (Figure 3c). The number of CD8+ T cells was approximately 10-fold higher in SCC FAK(- / -) tumors, implying an activated T cell response (Figure 3d), and GzmB, measured by ELISA, also showed high expression levels (Figure 3j). To assess whether cancer cells in SCC FAK(- / -) tumors contain intracellular activated GzmB, tumors were harvested and treated with probe H5 for 30 min before analysis by flow cytometry. Notably, more than 40% of cancer cells in SCC FAK(- / -) tumors were stained with probe H5, whereas wild-type SCC tumors were barely stained (Figures 3e, 3f). Furthermore, we observed that the fluorescent signal of probe H5 was found only in a subset of cancer cells, but not in other cells (e.g., monocytes, fibroblasts, CD4+ T cells) found in the tumor (Figures 3g, 3h, and 3k). Overall, these results support the utility of probe H5 for rapidly detecting T cell-mediated cell death in mouse tumors.

[0087] Probe H5 identifies immunomodulatory activity in drug screens and human tumor biopsies We next evaluated the utility of probe H5 in screening for immunomodulatory drugs and in clinical assays in human tumor biopsies (Figure 4a). First, we adapted a co-culture system of mouse CD8+ T cells and E0771 cancer cells to a 384-well plate format for an image-based phenotypic assay capable of screening small molecules for activating the killing capacity of CD8+ T cells. Using the ImageXpress high content analysis system, we tested a collection of anticancer drugs with different pharmacological targets (Table 3). E0771 and CD8+ T cells were treated with IL-2 (100 U mL -1 ) and co-cultured for 2 days before incubation with the individual drugs at their respective working concentrations (Table 3). One hour before imaging, probe H5 was added to the wells and fluorescent microscopy images were obtained with Hoechst33342 as a nuclear counterstain. The fluorescence intensity of probe H5 in the cancer cells was used to compare the immunomodulatory potential of all 44 drugs. A high concentration of IL-2 (250 U mL -1 ) as a negative control for low GzmB activity, and 100 U mL -1 In addition to IL-2, wells containing rapamycin, a known mTOR inhibitor that blocks IL-2-induced activation of T cells, were included. Notably, a small set of compounds with different pharmacological functions (e.g., protein kinase inhibitors, microtubule function inhibitors, DNA alkylating agents, proteasome inhibitors) showed superior staining to IL-2 treatment alone, demonstrating their ability to activate anticancer T cell activity.

[0088] Increased fluorescent signals (compared to IL-2 only controls) were observed in combination with IL-2 and different small molecules. Among these, compounds with different mechanisms of action were identified. AZD5363 (C5) is a protein kinase AKT inhibitor; docetaxel (C11), ARQ-621 (C12) and epothilone B (C13) are direct inhibitors of microtubule function; mitomycin C (C18) and temozolomide (C20) are DNA alkylating agents, and lactacystin (C30) is an irreversible proteasome inhibitor. Some of these compounds are already approved for medical use as anticancer drugs. Docetaxel (Taxotere®), mitomycin C (Mutamicin®) and temozolomide (Temodar®) are established chemotherapy drugs for the treatment of several types of cancer, including breast cancer (docetaxel), lung cancer (mitomycin) and brain cancer (temozolomide). Other compounds are currently being tested in clinical trials. For example, AZD5363, alone or in combination with other drugs (e.g., paclitaxel), is in Phase II trials in patients with metastatic breast or gynecologic cancers. ARQ621 is being evaluated in Phase I trials in patients with advanced solid or hematological malignancies. Similarly, epothilone B is in Phase II trials, particularly in patients with advanced colorectal, renal and prostate cancers.

[0089] Among these, the AKT kinase inhibitor AZD5363 showed the brightest H5 fluorescent staining and was selected for further study. We analyzed cell viability and the extent of H5 fluorescent staining in E0771 cancer cells incubated with AZD5363, and in co-cultures of E0771 cells with CD8+ T cells incubated with IL-2 alone or IL-2 + AZD5363. Treatment with AZD5363 alone did not induce significant cancer cell death or cause H5 staining, whereas treatment with 100 U mL -1The same concentration of AZD5363 in combination with IL-2 caused significant cancer cell death and H5 fluorescence in CD8+ T cell-E0771 cell cocultures (Figure 10). These observations are consistent with recent reports suggesting that AKT inhibition prevents CD8+ T cell exhaustion and enhances cytolytic activity against target cells. These results highlight the potential of AZD5363 to activate the anticancer activity of CD8+ T cells when combined with IL-2 and demonstrate the effectiveness of the H5-based imaging screen platform for the identification of novel immunomodulatory drugs.

[0090] Finally, we investigated whether probe H5 could also monitor cytotoxic T cell function in biopsies from lung cancer patients as a way to screen for potential predisposition to anticancer treatment. For these experiments, paired (i.e., normal and cancer tissue, Figure 4c) tissue sections were obtained from untreated lung cancer patients undergoing surgical resection. To analyze whether probe H5 could detect differences in CD8+ T cell reactivation between healthy and cancerous areas, all samples were treated with probe H5 and rapidly analyzed by flow cytometry together with an antibody-based panel of epithelial and immune cell markers. As shown in Figure 4d, we found significant differences in the percentage of EpCAM+H5+ cells between healthy and cancerous tissues of all patients, indicating the activation status and cytotoxic activity of CD8+ T cells against lung epithelial cells in the tumor microenvironment. Further optimization studies are required to adapt probe H5 for the evaluation of immunotherapy in cancer patients. Overall, our results demonstrate that probe H5 can be used in vitro in high-throughput screening assays to facilitate the discovery of novel immunotherapeutic combinations as well as in clinical characterization of human tumor biopsies, opening new avenues for accelerating the development of personalized anticancer immunotherapies.

[0091] conclusion We describe the rational design of fluorescent probes for the rapid detection and imaging of activated mouse and human GzmB. Starting from the common IEPD sequence, we demonstrated unprecedented cat / K M Ratio 1.2×10 7 M-1 s -1 We constructed a collection of FRET probes to optimize the hexapeptide H5 with a 6 pM detection limit and a 6 pM detection limit. We used molecular dynamics simulations to explore the binding mode of the peptide and observed that H5 binds to an extension of the catalytic groove defined by loop 74-84 and β-barrel 35-114 of hGzmB that is inaccessible by the tetrapeptide sequence. The discovery of this alternative binding mode for activated hGzmB may facilitate the design and optimization of imaging probes and enzyme inhibitors. We demonstrated that the fluorescence emission of H5 can be used as a direct reporter of immune-mediated tumor killing in live cultures of CD8+ T cells and cancer cells, both qualitatively by fluorescence microscopy and quantitatively by flow cytometry. Importantly, the probe H5 does not fluoresce in T cells where GzmB is inactive or in cancer cells killed by other agents not associated with immune-mediated cancer cell death (i.e., staurosporine). We also showed that probe H5 can identify mouse tumors undergoing immune-mediated regression in a model of squamous cell carcinoma and identify small molecule agents that activate immune-modulatory responses in an image-based phenotypic screen.Finally, we used H5 for the clinical characterization of human tumor biopsies, highlighting its potential application for personalized detection of early responses to anticancer immunotherapy.

[0092] Near-infrared granzyme B probe synthesis To further develop granzyme B technology, we sought to develop a near-infrared (NIR) emitting smart probe to detect active granzyme B. The use of NIR light exhibits several advantages over shorter wavelength emission, such as deeper penetration through the skin, reduced autofluorescence and minimal light scattering, enhancing the signal to noise in biological samples.

[0093] Based on the above mentioned work in developing GzmB specific sequences, we intended to retain a particularly effective hexapeptide sequence that confers rapid reactivity and selectivity with GzmB (Ile-Glu-Pro-Asp-Ala-Leu), as seen in probe H5. To produce NIR GzmB smart probes, it is necessary to exchange not only the fluorophore but also the quencher group in order to preserve the FRET process that renders the probe non-fluorescent in the absence of GzmB. Since changes in the smart probe components can reduce reactivity with the desired target, we attempted to minimize this risk by developing 12 NIR emitting smart probes using combinations of two different NIR fluorophores (silicon rhodamine and sulfo-Cy5), two linker types (hydrophobic and hydrophilic) and three different NIR quencher groups (QSY21, BXL-670 and BHQ-3). The chemical structures are shown below (note that the chemical structure of QXL-670 is not disclosed and is not available):

[0094] [ka] Chemical structures of fluorophores, linkers, and quenchers (except QXL-670) used in the development of NIR probes for detecting granzyme B activity.

[0095] The synthetic strategy for the NIR smart probe begins with solid-phase peptide synthesis on 2-chlorotrityl-linked polystyrene resin, where Asp (O t Bu) and Glu(O t Bu) with Fmoc / t Butyl strategy was adopted. tBu side chain protecting groups were utilized for orthogonality with Fmoc deprotection and peptide cleavage from the resin. Each coupling utilized COMU and Oxyma as coupling reagents, and 20% piperidine in DMF solution, 1M Oxyma for Fmoc removal (Oxyma was added to minimize aspartimide by-product formation during Fmoc removal). Each of the two fluorophores (see above) was coupled last before removal of the peptide from the resin with TFA (1% in DCM), rendering SiRho and SulfoCy5.

[0096] [ka] Solid-phase peptide synthesis of silicon rhodamine and SulfoCy5 containing granzyme B hexapeptide.

[0097] Next, a linker was incorporated at the free C-terminus of the peptide. Two linkers were selected for utilization: N-Boc-1,6-hexanediamine and tert-butyl (14 amino-3,6,9,12 tetraoxatetradecyl)carbamate. Each of these linkers exhibits contrasting polarity and were chosen to monitor their effect on substrate cleavage by GzmB. They both have side chain t The peptides were protected with mono-boc to release the terminal amine after butyl removal, and boc removal also requires a high percentage of TFA. Linker attachment was achieved by reacting the free C-terminus of the peptide with the respective linker in the presence of PyOxim, Oxyma, and DIPEA in a DCM / DMF (1:1) mixture at -20°C for 1.5 h. The crude mixtures were purified by reversed-phase semi-preparative HPLC.

[0098] [ka] Linker attachment to fluorophore peptide reagents using PyOxim and Oxyma as coupling reagents.

[0099] The next step in the sequence involved the removal of the t-butyl protecting groups from the glutamic and aspartic acid side chains as well as the boc-protected amines for all of the above probes. This was accomplished by dissolving the probes in DCM and treating with TFA (50%) for 1 h at room temperature. The solvent was then removed under reduced pressure and the remaining amorphous solid was purified by washing with diethyl ether.

[0100] [ka] Deprotection with TFA (40%) in DCM for all fluorophore-peptide linker conjugates

[0101] The final step in the synthesis of the complete NIR probes was the addition of the quencher moiety. Each quencher was purchased as a succinimide ester to allow for conjugation to the terminal amine of the linker after deprotection. The succinimide esters were sufficiently activated to not require a coupling reagent to form an amide bond with the terminal amine, thus avoiding potential side reactions with side chain carboxylic acid groups of the peptide sequence. To each of the fluorophore-peptide linker conjugates, each of the three quenchers (QXL-670, QSY21 or BHQ-3) was added in a separate reaction. DIPEA was then added to promote amide bond formation, and the reactions were stirred at room temperature for 2 days. After completion of the reactions (as determined by HPLC), the crude was purified by reverse-phase semi-preparative HPLC to give the final probes as blue amorphous solids (Table 4).

[0102] [ka] Addition of a quencher to the fluorophore-peptide-linker complex.

[0103] Reactivity of NIR probes with granzyme B These probes (Table 4) were used to test reactivity with human recombinant granzyme B and identify lead candidates. Each probe was tested at a concentration of 25 μM against 20 nM granzyme B and fluorescence was recorded over 2 hours at 37° C. (λ ex : 620nm, λ em : 660 nm). As a result, probe R7 showed the best performance, and its fluorescence intensity at 660 nm was increased by about 80-fold after the addition of granzyme B (Figure 14A). HPLC-MS was used to confirm the reactivity of R7 with granzyme B, showing that the conversion from intact to cleaved was 100% complete within 2 h, and the probe was cleaved after the aspartic acid residue (Figure 14B). Next, the selectivity of R7 for granzyme B was examined by incubating R7 with closely related proteases, namely -caspase-3 and granzyme A. No increase in fluorescence at 660 nm was observed, while preincubation of granzyme B with a known commercial inhibitor (Ac-IEPD-CHO) reduced the fold change, further confirming the specificity of R7 for granzyme B (Figure 14C). Finally, the kinetics of R7 consumption by human recombinant granzyme B was investigated by varying the substrate concentration and calculating the initial velocity at a fixed concentration of enzyme (20 nM) (kinetics at 25 μM substrate shown in FIG. 14D). These results indicate that R7 is approximately 1.98×10 6 M -1 s -1 K cat / K M It was stipulated that the

[0104] Optimization of assay conditions (with probe H5) To establish optimal probe settings, various concentrations of hGzmB (0, 8.4, 28, 84, 280, 840, 2800, 8400, 28000 and 84000 pg / ml) were used to compare the performance of probe H5 in different buffer compositions and optical settings. Unless otherwise stated in Table 5, 25 μM H5 was mixed with hGzmB in a 384-well black microplate and incubated at 37 °C before quantifying the fluorescence intensity. Probe concentration, ionic strength and pH of the buffer, as well as optical settings on the Synergy H1 Biotek microplate reader were varied to obtain the lowest possible LoD (Table 5).

[0105] Antibody capture activity assay (with probe H5) Because antibodies are highly specific for their substrates and are commonly used in immunoassay techniques to capture material from samples (especially those with complex matrices), an additional antibody capture step was added to detect GzmB in buffers and biological samples (e.g., serum). High-binding microplates (Merck) were coated with anti-GzmB antibodies in coating buffer and incubated overnight at 4°C in a humidified chamber. The plates were washed four times with wash buffer (0.01M PBS, 0.05% (v / v) Tween 20, pH 7.5) and 100ul of activated GzmB standards diluted to various concentrations in GzmB buffer (NaCl 100mM, Tris 50Mm, pH 7) from a 1mg / ml stock were added and incubated for 1 hour with shaking at 50 rpm. Samples were washed again 4 times with wash buffer and 100ul 25uM probe H5 diluted from a 10mM stock solution in optimized GzmB buffer was added and λ was measured at 0, 2, 4 and 24 hours using a Synergy H1 Biotek microplate reader. ex = 470 nm and λ emFluorescence intensity at 510 nm was read using a monochromator. Fluorescence intensity was plotted against GzmB concentration and the limit of detection (LoD) was calculated based on the mean + 3 standard deviations of the 0 concentration samples. Other ligands (e.g., proteins, nanobodies) can be used to capture the enzyme to the surface of the microwell plate as described above.

[0106] The LoD obtained in serum was 40.9 pg / ml, which is comparable to pathological levels of GzmB in blood and therefore has excellent potential for use in clinical samples.

[0107] Testing urine samples for acute transplant rejection background The gold standard test for detecting acute transplant rejection is to take a sample of material from the transplanted organ, called a core biopsy. However, this is an invasive test that can harm the patient, and one in ten cases does not provide enough tissue for a diagnosis. Granzyme B (GzmB) is a protein associated with acute transplant rejection, and measuring its activity can identify the early stages of rejection with high accuracy.

[0108] Kidney transplantation remains the single most effective treatment for end-stage renal disease. Approximately one in five patients will experience acute rejection within the first year of receiving a kidney transplant, which is associated with an increased risk of graft loss and death. Therefore, early detection and effective treatment of acute transplant rejection are critical to maximize graft function and quality of life for patients.

[0109] GzmB may be present in urine, but current methods for measuring it require expensive equipment and sample processing. Here, we address this issue by applying probes H5 and R7 to urine samples spiked with recombinant granzyme B to detect enzymatic activity.

[0110] result First, we investigated the stability of probes H5 and R7, and their stability in urine from three healthy donors to ensure that no cross-reactivity occurred with other biomolecules that may be present in all urine samples. To do this, we monitored the fluorescence response of probes H5 and R7 in urine with and without spiked granzyme B (15 nM) and also assessed their stability by HPLC analysis. Although the results showed a 7-fold increase in fluorescence at 510 nm for probe H5 and a 90-fold increase in fluorescence at 660 nm for probe R7, HPLC analysis revealed that less than 1% of both probes were cleaved only in urine samples, and up to 84% and 99% conversion of H5 and R7 (respectively) to the cleaved probes occurred after 1 h in the presence of granzyme B (Figure 11). We confirmed that probes H5 and R7 were cleaved after the aspartic acid residue (the native granzyme B cleavage site) by HPLC-MS.

[0111] Next, we investigated the detection of spiked recombinant granzyme B in urine samples from three healthy subjects to determine the limit of detection. A dynamic range of concentrations of activated recombinant granzyme B (20,000-78 pg / mL) was added to urine samples from healthy subjects, and the samples were incubated with probes H5 and R7 (25 μM) for 3 h at 37 °C. The LoD of probe H5 was 6.18 pM (198 pg / mL), and the LoD of probe R7 was 3.1 pM (99 pg / mL).

[0112] Examination of granzyme B activity in stool samples from IBD patients background Inflammatory bowel disease (IBD) is a chronic immune-mediated disease that causes inflammation of the digestive tract. During the last decade, IBD has emerged as a public health challenge, affecting 3 million people in the EU and with a global prevalence of over 0.3%. The most common cases are Crohn's disease and ulcerative colitis (UC).

[0113] Despite advances in IBD diagnosis and treatment, new biomarkers for IBD are needed to refine methods for monitoring intestinal inflammation, response to treatment, and choice of therapy. Current monitoring of IBD is performed by colonoscopy (an invasive procedure with a waiting time of several months), histological analysis of biopsies, or measuring the amount of limited proteins in blood or stool samples in clinics with results available in 2–3 weeks. Colonoscopy is invasive and expensive; whereas imaging scans (e.g., magnetic resonance imaging (MRI), computed tomography (CT)) do not provide a direct assessment of the intestinal mucosa. Mucosal healing has been used to assess the efficacy of treatment in IBD patients, but is heavily dependent on histology from invasive biopsies over time.

[0114] Some biomarkers for IBD already exist, such as C-reactive protein and calprotectin, which can be detected in blood / stool samples. However, these biomarkers are general indicators of inflammation and cannot specifically report on IBD activity. It is therefore desirable to have additional or more specific markers at the clinician's disposal.

[0115] Granzyme B is directly involved in inflammatory diseases and is highly active during chronic intestinal inflammation, as shown in a study with serum from IBD patients (R. Kalla et al., J. Crohn's. Colitis. 2020, 15, 699-708) and in intestinal tissue biopsies. Very high levels of activated granzyme B damage the intestine and are one of the reasons why inflammation does not disappear. The intestine of IBD patients has a high infiltration of immune cells (T cells) that can induce intestinal damage in a granzyme B-dependent manner. The development of the first IVD for granzyme B with our fluorescent chemical probe could revolutionize the current process of IBD diagnosis and monitoring via colonoscopy. Here, we utilize probes H5 and R7 in clinical stool samples based on calprotectin levels to establish whether granzyme B activity can also distinguish between health and disease.

[0116] result In this study, we used a proprietary antibody capture assay to evaluate granzyme B activity in each stool sample (Figure 12). First, high-binding 96-well plates were coated with granzyme B antibody for 2 h at 37 °C, the bound antibody was blocked using Chonblock (2 h at room temperature), washed, and clinical stool samples were added and incubated at room temperature for 2 h. The plates were then washed, probes H5 and R7 were added, and fluorescence was monitored for 18 h at 37 °C. To quantify the detected granzyme B signal, a calibration curve was set up on each 96-well plate, and known concentrations of human recombinant granzyme B were added to each well.

[0117] Forty-eight 'high calprotectin' and 46 'low calprotectin' stool supernatants were analysed and the fluorescence fold change of probes H5 and R7 was compared. The fold change was quantified to obtain concentration values ​​along a standard curve (Figure 13A and B). When quantified, the mean concentrations of low and high calprotectin samples using probe H5 were 4802 pg / mL and 11636 pg / mL, respectively (Figure 13A). On the other hand, the mean concentrations of low and high calprotectin samples using probe R7 were 3318 pg / mL and 5687 pg / mL, respectively (Figure 13B).

[0118] [Table 1]

[0119] [Table 2]

[0120] [Table 3] TIFF2024527471000014.tif152170

[0121] [Table 4]

[0122]

Table 5

[0123] 参考文献 1. Mouchacca, P., Schmitt-Verhulst, A. M., & Boyer, C. Visualization of cytolytic T cell differentiation and granule exocytosis with t cells from mice expressing active fluorescent granzyme B. PLoS One 8, e67239(2013). 2. Thornberry, N. A. et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B: functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17907 - 17911(1997). 3. Mac, Q. D. et al. Non-invasive early detection of acute transplant rejection via nanosensors of granzyme B activity. Nat. Biomed. Eng. 3, 281 - 291(2019). 4.He, S., Li, J., Lyu, Y., Huang, J. & Pu, K. Near-infrared fluorescent macromolecular reporters for real-time imaging and urinalysis of cancer immunotherapy. J. Am. Chem. Soc. 142, 7075-7082(2020). 5.Espargaro, A. et al. On the binding of Congo Red to amyloid fibrils. Angew. Chem. Int. Ed. 59, 8104-8107(2020). 6.Gotz, A. W. et al. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 1. generalized born. J. Chem. Theory. Comput. 8, 1542-1555(2012). 7.Serrels, A. et al. Nuclear FAK controls chemokine transcription, Tregs, and evasion of anti-tumor immunity. Cell 163, 160-173(2015). 8.Janiszewski, T. et al. Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes. J. Biol. Chem. 295, 9567-9582(2020). 9.Mahrus, S. & Craik, C. S. Selective chemical functional probes of granzymes A and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells. Chem. Biol. 12, 567-577(2005). 10.Harris, J. L., Peterson, E. P., Hudig, D., Thornberry, N. A. & Craik, C. S. Definition and redesign of the extended substrate specificity of granzyme B. J. Biol. Chem. 273, 27364-27373(1998). 11.Kok, H. M. et al. Systemic and local granzyme B levels are associated with disease activity, kidney damage and interferon signature in systemic lupus erythematosus. Rheumatology 56, 2129-2134(2017). 12.Canel, M., Taggart, D., Sims, A. H., Lonergan, D. W., Waizenegger, I. C. & Serrels, A. T-cell co-stimulation in combination with targeting FAK drives enhanced anti-tumor immunity. eLife 9, e48092(2020).

Claims

1. A probe for use in detecting granzyme B, comprising a granzyme B-cleavable peptide linked or conjugated to a detectable moiety, wherein the sequence of the granzyme B-cleavable peptide is the following sequence IEPDAL consisting of, a probe.

2. The probe according to claim 1, wherein the detectable moiety is an isotope label or a radiation label, a paramagnetic contrast agent, paramagnetic or superparamagnetic particles, or an optically detectable moiety.

3. The probe according to claim 2, wherein the optically detectable moiety is a fluorescent moiety.

4. The probe according to claim 3, further comprising a quencher moiety.

5. The probe according to claim 4, wherein the detectable moiety and / or the quencher moiety is linked to the peptide via a covalent bond.

6. The probe according to claim 4, wherein the detectable moiety is linked to the N or C terminal amino acid of the peptide, and the fluorescent moiety is linked to the respective C or N terminal to which it is linked.

7. The probe according to claim 4, wherein the detectable moiety, the fluorescent moiety and / or the quencher moiety is linked, conjugated and / or embedded within a nanoparticle, and the nanoparticle is linked to the peptide.

8. The probe according to claim 7, wherein the detectable moiety, the fluorescent moiety, the quencher moiety and / or the nanoparticle is linked to the peptide via a linker molecule.

9. The probe according to claim 8, wherein the linker molecule is an alkyl, alkenyl, or polyether chain, optionally having C2-C24 repeating units, or one or more natural or unnatural amino acids or imino acids.

10. The probe according to claim 1, which is capable of detecting granzyme B in a sample in an amount less than 10 nM, such as less than 1 nM, less than 500 pM, less than 250 pM, less than 100 pM, or less than 50 pM.

11. The probe according to claim 1, wherein the Km of granzyme B is less than 30 μM, less than 25 μM, less than 20 μM, less than 15 μM, or less than 10 μM.

12. The kcat / KM value is 1×10 ４ greater, 1×10 ５ greater, 1×10 ６ greater, or 1×10 ７ greater than that of the probe according to claim 1.

13. A detection device comprising the probe according to any one of claims 1 to 12, wherein the probe is bound, adhered, or otherwise captured on the surface of the device.

14. The detection device according to claim 13, wherein the surface is the surface of a slide, a microtiter plate, a lateral flow device, or the wall of a fluid device.

15. The detection device according to claim 13, wherein the surface is the surface of beads, microparticles, or nanoparticles.

16. Use of the probe according to any one of claims 1 to 12 in the ex vivo or in vitro detection of the level of granzyme B in a cell and / or body fluid sample.

17. A method for detecting granzyme B in a cell-containing sample or body fluid or excrement, comprising contacting the probe according to any one of claims 1 to 12 with a cell-containing sample or body fluid / excrement sample or a processed part thereof ex vivo, in vivo, or in vitro, and detecting the level of granzyme B by cleavage of the peptide and release of the cleaved peptide containing the detectable part.

18. The method according to claim 17, wherein the body fluid is a blood, serum or plasma sample.

19. The method according to claim 17, wherein the body fluid is urine or a lung lavage fluid.

20. The method according to claim 17, wherein the body fluid / excrement is a sample such as a fecal or urine sample that has been treated to concentrate and / or separate granzyme B from other components in the sample.

21. The treated sample is contacted with a binding agent such as an anti-granzyme B antibody or a binding fragment thereof that can specifically bind to granzyme B, and optionally the binding agent is bound, adhered, or otherwise attached to a surface before contacting the probe to detect the level of granzyme B present in the diluted fecal sample. The method according to claim 20.

22. A composition for detecting granzyme B, the composition comprising the probe according to any one of claims 1 to 12.

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