Protease activity sensors and methods of using the same

Description

Attorney Docket No.10034-321WO1 PROTEASE ACTIVITY SENSORS AND METHODS OF USING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 708,471, filed October 17, 2024, which is incorporated by reference herein in its entirety. GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under CA265711 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND

[0003] Advances in synthetic biology have historically focused on the genetic circuit paradigm using parts such as inducible promoters, activators, and repressors to assemble sense- and-respond biocircuits that operate under transcriptional regulation. Increasingly sophisticated functions have been successfully implemented in a variety of prokaryotic and eukaryotic cells with far ranging applications in biomedicine to enhance cell therapies, drug delivery, molecular imaging, and biosensors. For example, bacteria have been genetically engineered to report on human disease either by detecting clinically useful biomarkers or producing synthetic biomarkers in biofluids including human urine and serum. In mammalian cell engineering, sense-and-respond components that employ AND, OR, or NOT gate logic have been demonstrated for multiplexed and programmable control of T cell responses, thereby increasing the specificity of tumor-antigen sensing and reducing systemic toxicity. SUMMARY

[0004] In some aspects, disclosed herein is a protease activity sensor including the sequence: X3-X1-k-R1-kpP-X4-k-X5-R2-k-X2-K-X6(SEQ ID NO: 1), wherein: k is D-lysine; p is D-proline; X1and X2are amino acids crosslinked by their side chains; X3is a quencher or an acetyl group and X4is a reporter molecule, or X3is a reporter molecule and X4is null or a quencher; X5is null or cysteine-D-lysine (Ck); X6is glycinamide; and R1and R2are protease cleavage sites for the same or different proteases. ^Attorney Docket No.10034-321WO1

[0005] In some aspects, also disclosed herein is a nanoparticle including a plurality of any of the disclosed protease activity sensors anchored to a surface of said nanoparticle.

[0006] In some aspects, also disclosed herein is a method of detecting presence of a protease, the method including: a) providing any of the disclosed protease activity sensors or any of the disclosed nanoparticles to a sample or a subject, wherein R1and R2are protease cleavage sites for the same protease; and b) detecting presence or absence of the reporter molecule in the sample, the subject, or a sample taken from the subject, thereby detecting presence or absence of the protease; wherein presence of the reporter molecule indicates presence of the protease; and wherein absence of the reporter molecule indicates absence of the protease.

[0007] In some aspects, also disclosed herein is a method of detecting presence of two proteases, the method including: a) providing any of the disclosed protease activity sensors or any of the disclosed nanoparticles to a sample or a subject, wherein R1and R2are protease cleavage sites for different proteases; and b) detecting presence or absence of the reporter molecule in the sample, the subject, or a sample taken from the subject, thereby detecting presence or absence of the two proteases; wherein presence of the reporter molecule indicates presence of both of the two proteases; and wherein absence of the reporter molecule indicates absence of one or both of the two proteases.

[0008] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims. BRIEF DESCRIPTION OF DRAWINGS

[0009] FIGURES 1A-1F depict that asymmetric bi-labile cyclic peptide nanosensors implement AND-gate logic. FIG. 1A shows the design of AND-gated nanosensors, including iron oxide nanoparticles decorated with asymmetric bi-labile cyclic peptides that release a fluorescent reporter only when both substrates are cleaved, thereby actuating AND-gate logic. FIGS.1B-1C show fluorescence upon treatment of quenched linear substrates for granzyme B (FIG.1B, GzmB; substrate: IEFDSG (SEQ ID NO: 10)) and matrix metalloproteinases (FIG. 1C, MMP; substrate: PAALRA (SEQ ID NO: 14)) with recombinant GzmB or MMP8 for 15 minutes (one-way analysis of variance (ANOVA) with Dunnett’s post-test and correction for multiple comparisons). FIG.1D shows mass spectral analysis by MALDI-MS (matrix-assisted laser desorption ionization mass spectrometry) of an asymmetric bi-labile cyclic peptide ^Attorney Docket No.10034-321WO1 bearing substrates for GzmB and MMPs (i.e., an AND-gated peptide) confirms production of a cleaved reporter fragment (1407.6 Da) only after treatment with both GzmB and MMP8. FIGS.1E-1F show a representative kinetic curve (FIG.1E) and fluorescence at 2 hours (FIG. 1F) for quenched fluorescent AND-gated nanosensors treated with GzmB and / or a panel of MMPs (FIG. 1E uses two-way ANOVA with Dunnett’s post-test and correction for multiple comparisons; FIG. 1F uses one-way ANOVA with Tukey’s post-test and correction for multiple comparisons). (FIGS. 1A-1F use ***P < 0.001, ****P < 0.0001, n = 3 technically independent wells, error bars depict mean ± s.e.m.; ns, not significant; RFU, relative fluorescent units).

[0010] FIGURES 2A-2B depict synthesis of bi-labile cyclic peptides. FIG.2A shows the structure of a prototypical bi-labile cyclic peptide for AND-gated sensing. FIG. 2B shows a summary of synthetic scheme of quenched fluorescent bi-labile cyclic peptides, with confirmation by ESI-MS (electrospray ionization mass spectrometry). A linear bi-labile peptide is synthesized by solid phase peptide synthesis and cyclized by lactamization between a side chain amine and carboxylate on lysine and glutamate residues. FRET (Förster resonance energy transfer) pair labeling is performed by reaction of activated ester of dye (in prototypic example, 5(6)-carboxyfluorescein (5(6)-FAM) fluorophore) to N-terminal amine and copper- catalyzed azide-alkyne cycloaddition of azide-functionalized dye (in prototypic example, Tide Quencher 2 (TQ2) quencher) to L-propargylglycine side chain alkyne. Sequences in FIGS. 2A-2B: IEFDSG (SEQ ID NO: 10), Z-EkIEFDSGkpPXkIEFDSGkKKG (SEQ ID NO: 18).

[0011] FIGURES 3A-3F depict that cyclic peptides require cleavage of both substrates for reporter release. FIG. 3A shows a kinetic curve of fluorescence upon GzmB cleavage of quenched fluorescent linear GzmB substrate (two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIG. 3B shows mass spectral analysis by ESI-MS of linear GzmB substrate treated with GzmB. FIGS.3C-3D show a kinetic curve of fluorescence (FIG.3C) and ESI-MS mass spectral analysis (FIG.3D) for GzmB cleavage of symmetric bi- labile cyclic peptide bearing two copies of GzmB substrate (FIG. 3C uses two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIG. 3E shows fluorescence upon GzmB cleavage of linear GzmB substrate and non-labile control substrate synthesized using d-amino acids (two-tailed Student’s t-test. FIG. 3F shows fluorescence upon GzmB cleavage of fully labile cyclic peptide and two control peptides where one of the substrates is non-labile due to the incorporation of d-amino acids (one-way ANOVA with Dunnett’s post- test and correction for multiple comparisons). (FIGS. 3A-3F use ****P < 0.0001, n = 3 technically independent wells, error bars depict mean ± s.e.m.). ^Attorney Docket No.10034-321WO1

[0012] FIGURES 4A-4B depict cleavage of MMP substrate by matrix metalloproteinases. FIG. 4A shows fluorescence upon treatment of quenched fluorescent linear MMP substrate with various collagenases and gelatinases. Cleavage is abrogated in the presence of marimastat, an MMP inhibitor. FIG.4B shows cleavage fluorescence measured 30 minutes after incubation of fluorogenic PAALRA (SEQ ID NO: 14) substrate (5 ^M) with the indicated recombinant protease (50 nM). (FIG.4A uses one-way ANOVA with Dunnett’s post-test and correction for multiple comparisons, *P = 0.0149, ****P < 0.0001, n = 3 technically independent wells, error bars depict mean ± s.e.m.; ns, not significant).

[0013] FIGURES 5A-5B depict AND-gated sensing of granzyme B and matrix metalloproteinases using cyclic peptides. FIGS. 5A-5B show a representative kinetic curve (FIG. 5A) and fluorescence (FIG. 5B) upon treatment of AND-gated peptide with GzmB and / or MMPs. (one-way ANOVA with Tukey’s post-test and correction for multiple comparisons, ****P < 0.0001, n = 3 technically independent wells, error bars depict mean ± s.e.m.).

[0014] FIGURES 6A-6C depict synthesis and characterization of AND-gated nanosensors. FIG.6A shows a schematic of AND-gated nanosensor formulated by conjugation of iron oxide nanoparticle (IONPs; containing free surface amines) to bi-labile cyclic peptides (containing free side chain thiol) using SM(PEG)6 heterobifunctional crosslinker (containing succinimidyl ester and maleimide). FIGS. 6B-6C show number average size distribution determined by dynamic light scattering (FIG. 6B) and absorbance spectrum determined by ultraviolet-visible (UV-Vis) spectrophotometry (FIG. 6C) for bare IONPs and AND-gated nanosensors.

[0015] FIGURES 7A-7C depict that modular design enables AND-gated sensing of granzyme B and thrombin. FIGS.7A-7B show fluorescence upon treatment of quenched linear substrates for GzmB (FIG.7A) and thrombin (FIG.7B, Thrb; substrate: fPRSG (SEQ ID NO: 15) with recombinant GzmB or Thrb (one-way ANOVA with Dunnett’s post-test and correction for multiple comparisons). FIG. 7C shows a kinetic curve of AND-gated nanosensor, designed with bi-labile cyclic peptides containing the GzmB substrate and fPRSG (SEQ ID NO: 15), upon cleavage by GzmB and / or Thrb. (a-c, ***P < 0.001, ****P < 0.0001, n = 3 technically independent wells, error bars depict mean ± s.e.m.; ns, not significant). Sequences in FIGS.7A-7C: IEFDSG (SEQ ID NO: 10), fPRSG (SEQ ID NO: 15).

[0016] FIGURES 8A-8C depict extending modularity of AND-gated nanosensors. FIG. 8A shows a heat map of initial cleavage velocity upon treatment of four different linear peptide FRET substrates with MMP9, GzmB, fibroblast activation protein (FAP), or legumain (LGN). ^Attorney Docket No.10034-321WO1 FIGS. 8B-8C show fluorescence of AND-gated nanosensor, designed with bi-labile cyclic peptides containing the MMP substrate and either AAN (SEQ ID NO: 12) (LGN substrate, FIG. 8B) or ASGPAGPA (SEQ ID NO: 13) (FAP substrate, FIG. 8C), upon cleavage by MMP9 and / or LGN or FAP. (FIGS. 8A-8C, n = 3 technically independent wells, error bars depict mean ± s.e.m.). Other sequences in FIGS.8A-8C: PAALRA (SEQ ID NO: 14).

[0017] FIGURES 9A-9G depict that multivalent presentation of AND-gated peptides improves proteolysis kinetics. FIGS.9A-9B show Michaelis-Menten analysis of rate of GzmB catalysis for linear and symmetric bi-labile cyclic peptides when free in solution (FIG.9A) or multivalently presented on iron oxide nanoparticles (FIG. 9B). FIG. 9C shows initial GzmB cleavage velocity for nanoparticles (NPs) labeled with varying numbers of symmetric bi-labile cyclic peptides per NP. FIGS. 9D-9E show dose dependence of GzmB-mediated cleavage fluorescence for nanosensors displaying linear (FIG.9D) and symmetric bi-labile cyclic (FIG. 9E) peptides. Dashed line indicates detection of statistically significant signal relative to blank. FIGS. 9F-9G show cleavage fluorescence (FIG. 9F) and Michaelis-Menten analysis (FIG. 9G) for AND-gated nanosensors displaying asymmetric bi-labile cyclic peptides when cleaved by varying concentrations of GzmB and / or MMP9. (FIGS. 9A-9G use n = 3 technically independent wells, error bars depict mean ± s.e.m.).

[0018] FIGURES 10A-10D depict quantification and cleavage kinetics of multivalent AND-gated nanosensors. FIG. 10A shows a formula for calculation of valency based on absorbance spectrum determined by UV-vis spectroscopy. FIG. 10B shows the effect of stoichiometric ratio of cyclic peptides used in crosslinking reaction on valency of cyclic peptides conjugated to nanosensors. Valency varies linearly with a broad range of stoichiometric ratios. FIGS.10C-10D show kinetic curves for GzmB cleavage of nanosensors displaying varying numbers of symmetric bi-labile cyclic peptides. (FIGS.10C-10D use n = 3 technically independent wells, error bars depict mean ± s.e.m.).

[0019] FIGURES 11A-11F depict activation of AND-gated peptides by co-localized proteases from T cells and cancer cells. FIG. 11A shows a schematic for collection of conditioned media containing cell-secreted proteases, including GzmB from CD8 T cells activated by ^CD3 and ^CD28 antibodies or MMPs from MC38 cancer cells. FIGS.11B-11C show concentrations of GzmB (FIG. 11B) and MMP9 (FIG. 11C) in conditioned media determined by enzyme-linked immunosorbent assay (ELISA) (two-tailed Student’s t-test). FIGS.11D-11E show cleavage fluorescence of quenched fluorescent linear peptides for GzmB (FIG. 11D) and MMP (FIG. 11E) when treated with unconditioned media or media conditioned by the indicated cell (one-way ANOVA with Dunnett’s post-test and correction ^Attorney Docket No.10034-321WO1 for multiple comparisons). FIG. 11F shows cleavage fluorescence of asymmetric bi-labile cyclic peptide sensitive to GzmB and MMPs when treated with conditioned media from CD8 T cells and / or MC38 cancer cells (two-way ANOVA with Tukey’s post-test and correction for multiple comparisons). (FIGS.11A-11F use ***P < 0.001, ****P < 0.0001, n = 3 technically independent wells, error bars depict mean ± s.e.m.). Sequences in FIGS. 11A-11F: IEFDSG (SEQ ID NO: 10), PAALRA (SEQ ID NO: 14).

[0020] FIGURES 12A-12E depict that AND-gated nanosensors selectively report on T cell killing of cancer cells. FIG.12A is a schematic of co-culture assay using TCR-transgenic OT-1 T cells and MC38 cancer cells that were pulsed with the cognate OVA257-264antigen (SIINFEKL (SEQ ID NO: 16)) or a LCMV gp34-41 (AVYNFATC (SEQ ID NO: 17)) as a negative control peptide. FIG. 12B shows cytotoxicity quantified by release of lactate dehydrogenase (LDH) (one-way ANOVA with Tukey’s post-test and correction for multiple comparisons). FIGS. 12C-12D show concentrations of GzmB (FIG. 12C) and MMP9 (FIG. 12D) in co-culture media determined by ELISA (two-way ANOVA with Tukey’s post-test and correction for multiple comparisons). FIG. 12E shows cleavage fluorescence of AND-gated nanosensor when treated with co-culture media (two-way ANOVA with Dunnett’s post-test and correction for multiple comparisons). (FIGS.12A-12E use *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 3 biologically independent wells, error bars depict mean ± s.e.m.).

[0021] FIGURES 13A-13J depict that AND-gated nanosensors detect antitumor responses during immune checkpoint blockade therapy. FIG. 13A shows tumor growth kinetics for C57BL / 6J (B6) mice bearing subcutaneous MC38 tumors. Starting on day 13, mice were treated every 3 days for up to 4 doses of immune checkpoint blockade (ICB) therapy or isotype (Iso) control treatment. ICB therapy included intravenous administration of 0.2 milligrams each of ^PD1 and ^CTLA4 antibodies and intraperitoneal administration of 10 ^g of IL-2 cytokine. Iso control treatment included intravenous administration of 0.2 mg each of IgG1 and IgG2b isotype control antibodies (n = 7-8 biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIGS. 13B-13D show representative flow plots (FIG. 13B), frequency (FIG. 13C), and number per gram of tissue (FIG. 13D) of GzmB+CD8+ T cells isolated from MC38 tumors in mice treated with ICB or Iso. Flow analysis was performed one day after the third treatment dose (n = 5-7 biological replicates, Student’s t-test). FIGS. 13E-13G show a schematic (FIG. 13E), representative images (FIG. 13F), and quantification (FIG. 13G) of near-infrared fluorescence (NIRF) in MC38 tumors and major organs (liver, lungs, heart, spleen, kidneys, brain, and tumor-draining lymph nodes (tdLNs)) upon intratumoral (i.t.) administration of quenched NIRF AND-gated ^Attorney Docket No.10034-321WO1 nanosensors to tumor-bearing mice one day after the third ICB or Iso treatment dose. Images were taken 3 hours after sensor administration. Relative fluorescence in each organ (RFU of organ / total RFU from all excised organs for each mouse) was normalized to average signal for Iso-treated mice (n = 6-7 biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIGS. 13H-13J show a urinalysis schematic (FIG. 13H), quantification (FIG.13I), and receiver-operator characteristic (ROC) curves (FIG.13J) of carboxyfluorescein (5(6)-FAM) reporters in urine of tumor-bearing mice receiving i.t. administration of 5(6)-FAM-labeled AND-gated nanosensors one day after the third treatment. Urine was collected 3 hours after sensor administration. Reporters were purified from urine by immunoprecipitation and quantified by fluorescence, which was normalized to the average signal for Iso-treated mice at each timepoint. B2m– / –tumors were generated from MC38 cancer cells with knocked out expression of B2m (beta-2 microglobulin) (n = 3-8 biological replicates, two-way ANOVA with Tukey’s post-test and correction for multiple comparisons). (FIGS. 13A-13J use *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant; AUC, area under the ROC curve).

[0022] FIGURES 14A-14C depict individual tumor growth and survival curves for MC38 tumor-bearing mice treated with immune checkpoint blockade therapy. FIG. 14A depicts a schedule for treatment and analysis (flow cytometry or analysis of AND-gated sensors by NIRF imaging or urinalysis) of mouse models of ICB therapy using MC38 tumors. FIGS.14B-14C depict individual tumor growth curves (FIG. 14B) and survival curves (FIG. 14C) for MC38 tumor-bearing mice. Seven out of 8 mice treated with ICB demonstrated complete response (CR). (****P < 0.0001, n = 7-8 biological replicates, Mantel-Cox log-rank test).

[0023] FIGURE 15 depicts a gating strategy for flow cytometric analysis of CD8+ T cells in MC38 tumors.

[0024] FIGURES 16A-16B depict cleavage of near-infrared fluorescent AND-gated nanosensors by recombinant and cell-secreted proteases. FIG. 16A shows cleavage fluorescence of quenched NIRF AND-gated nanosensors when treated with GzmB and / or gelatinases (MMP2 or MMP9) (n = 3 technically independent wells, one-way ANOVA with Tukey’s post-test and correction for multiple comparisons). FIG.16B shows a kinetic curve of cleavage fluorescence of quenched NIRF AND-gated nanosensors when treated with conditioned media from co-culture of OT-1 T cells and / or MC38 tumors pulsed with the cognate OVA antigen (n = 3 biologically independent wells, two-way ANOVA with Dunnett’s post-test and correction for multiple comparisons). (FIGS.16A-16B use ****P < 0.0001). ^Attorney Docket No.10034-321WO1

[0025] FIGURES 17A-17B depict biodistribution of activated AND-gated nanosensors in tumor and major organs. FIGS.17A-17B show biodistribution of tissue fluorescence produced by NIRF AND-gated sensors administered to MC38 tumor-bearing mice given either Iso (FIG. 17A) or ICB (FIG. 17B) treatment. Fluorescence (RFU) is normalized to total fluorescence detected across all excised organs for each mouse. (FIGS. 17A-17B use n = 6-7 biological replicates).

[0026] FIGURES 18A-18B depict that AND-gated nanosensors differentiate ICB- responsive and resistant tumors. FIGS.18A-18B show representative images (FIG.18A) and quantification (FIG. 18B) of NIRF (RFU / area) in wild-type (w.t.) and B2m– / –MC38 tumors upon i.t. administration of quenched NIRF AND-gated nanosensors to tumor-bearing mice one day after the third ICB or Iso treatment dose. Images were taken 3 hours after sensor administration. (*P < 0.05, n = 6-8 biological replicates, one-way ANOVA with Dunnett’s post-test and correction for multiple comparisons).

[0027] FIGURES 19A-19B depict selective activation of AND-gated nanosensors in tumor lysates. Tumor and major organs were isolated from MC38 tumor-bearing mice and subjected to bead homogenization to produce lysate. FIG. 19A shows cleavage fluorescence measured 30 minutes after incubation of fluorogenic PAALRA (SEQ ID NO: 14) substrate (5 ^M) with homogenate from the indicated tissue. FIG.19B shows quenched NIRF AND-gated nanosensors were treated with a mixture of lysate and recombinant GzmB. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 3-12 biological replicates, one-way ANOVA with Dunnett’s post-test and correction for multiple comparisons; Br, brain; H, heart; Ki, kidneys; Li, liver; LN, lymph nodes; Lu, lungs; Spl, spleen; Tu, tumor).

[0028] FIGURES 20A-20I depict that AND-gated nanosensors increase specificity by requiring co-localized proteases for activation. FIG. 20A shows a schematic and kinetics of body weight for mouse model of viral infection using intranasal administration of PR8 influenza A (30 plaque-forming units) (n = 10 biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIGS. 20B-20C show representative flow plots (FIG.20B) and number (FIG.20C) of GzmB+CD8+ T cells isolated from bronchoalveolar lavage fluid (BALF) and lungs of naïve and PR8-infected mice eight days post-infection. Dump channel includes cells stained with ^CD4, ^CD19, and ^NK1.1 antibodies (n = 3 biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIG.20D shows a schematic of detection specificity for AND-gated sensor. A linear GzmB sensor may activate during both antiviral responses to influenza infection and antitumor responses to ICB therapy. By contrast, an AND-gated sensor may be ^Attorney Docket No.10034-321WO1 tumor-specific and remain inactive during antiviral responses. FIGS. 20E-20F show representative images (FIG.20E) and quantification (FIG.20F) of NIRF (RFU) in lungs upon intravenous (i.v.) administration of quenched NIRF linear GzmB nanosensors or AND-gated nanosensors to PR8-infected mice 8 days post-infection. Images were taken 3 hours after sensor administration (n = 5 biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIG. 20G shows a schematic of possible outcomes for AND-gated nanosensors in distal tumors after i.t. administration to a local tumor in a bilateral tumor model. If sensors were partially cleaved by GzmB in the local tumor, they would activate distally in both resistant B2m– / –and responsive wild-type (w.t.) tumors independent of GzmB expression in the distal tumor. By contrast, if sensors remained predominantly intact upon arrival to distal tumors, they would retain the ability to discriminate resistant and responsive tumors dependent on GzmB expression. FIGS. 20H-20I show representative images of local and distal tumors (FIG. 20H) and quantification of NIRF (RFU / area) in distal tumors (FIG. 20I) for bilateral tumors upon i.t. administration of quenched NIRF AND-gated nanosensors to local tumors (n = 6-8 biological replicates, one-way ANOVA with Dunnett’s post-test and correction for multiple comparisons). (FIGS. 20A-20I **P < 0.01, ****P < 0.0001; ns, not significant).

[0029] FIGURE 21 depicts individual body weight curves for mice infected with PR8 influenza A virus. (n = 10 biological replicates). FIG.21A shows body weight of naïve mice. FIG.21B shows body weight of mice infected with PR8.

[0030] FIGURES 22A-22B depict specificity of AND-gated nanosensors to GzmB activity in flu-infected lungs. FIGS.22A-22B show representative image and quantification of NIRF produced by i.v. administration of quenched fluorescent linear GzmB sensors (FIG. 22A) or AND-gated nanosensors (FIG.22B) in major organs (kidneys, liver, lungs, spleen) of naïve or PR8-infected mice. Fluorescence (RFU) for each organ was normalized to the respective average signal in naïve mice. (FIGS.22A-22B use n = 5 biological replicates, ****P < 0.0001, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons; ns, not significant).

[0031] FIGURES 23A-23G depict the toxicology of AND-gated nanosensors. FIG. 23A shows a treatment schedule including two doses of AND-gated nanosensors administered i.v., with serum analysis one week after the second dose. FIGS. 23B-23C show kinetics of body weight (FIG. 23B) and temperature (FIG. 23C). FIG. 23D shows the time course of body temperature immediately after the second dose. FIGS.23E-23G show analysis of major serum metabolites (FIG.23E), ions (FIG.23F), and cytokines (FIG.23G) at endpoint. In FIG.23G, ^Attorney Docket No.10034-321WO1 dashed line indicates limit of detection (LOD), and all values below LOD are plotted as zero; C refers to a control sample containing a mixture of recombinant proteins (n = 2 technical replicates). (FIGS.23B-23C use n = 10 biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons; FIGS. 23D-23G use n = 5 biological replicates, two-tailed Student’s t-test; ns, not significant).

[0032] FIGURES 24A-24B depict tissue fluorescence produced by intravenously administered linear GzmB nanosensors. FIGS. 24A-24B show a representative image (FIG. 24A) and quantification (FIG. 24B) of fluorescence in MC38 tumors and major organs produced by i.v. administration of NIRF linear GzmB nanosensors to tumor-bearing mice one day after the third ICB or Iso treatment dose. Images were taken 3 hours after sensor administration. Fluorescence (RFU / area) in each organ was normalized to average signal for Iso-treated mice. (n = 5-6 biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons; ns, not significant)

[0033] FIGURES 25A-25F depict that AND-gated nanosensors increase selectivity of on- tumor detection. FIGS.25A-25B show a representative image (FIG. 25A) and quantification (FIG. 25B) of fluorescence in MC38 tumors and major organs produced by intravenous (i.v.) administration of NIRF AND-gated nanosensors to tumor-bearing mice one day after the third ICB or Iso treatment dose. Images were taken 3 hours after sensor administration. Fluorescence (RFU / area) in each organ was normalized to average signal for Iso-treated mice (n = 6-7 biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIG.25C shows average tumor-to-organ selectivity for each organ produced by linear GzmB nanosensors or AND-gated nanosensors. Tumor-to-organ selectivity is defined as the ratio of isotype-normalized fluorescence in ICB-treated tumors compared to the corresponding normalized fluorescence in a respective non-tumor organ. Average selectivity was calculated using the mean from n = 6-7 biological replicates (two-tailed paired Student’s t-test). FIG. 25D shows quantification of fluorescence in MC38 tumors and major organs produced by i.v. administration of NIRF AND-gated nanosensors to tumor-bearing mice that were treated with one dose of oxaliplatin (0.1 mg, i.p.) followed by three doses of ICB or Iso. Fluorescence (RFU / area) in each organ was normalized to average signal for mice treated with Iso only (n = 4-7 biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIGS. 25E-25F show quantification of fluorescence in MC38 tumors, lungs, and other major organs (FIG.25E) and representative images (FIG.25F) after i.v. administration of NIRF AND-gated nanosensors to tumor-bearing mice infected with PR8 influenza A virus and treated with three doses of ICB or Iso. Images are displayed at same scale ^Attorney Docket No.10034-321WO1 as corresponding images of liver and spleen in FIG.27. Fluorescence (RFU / area) in each organ was normalized to average signal for uninfected control mice treated with Iso only (n = 5-8 biological replicates, two-way ANOVA with Dunnett’s post-test and correction for multiple comparisons). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ndLNs, non-tumor- draining lymph nodes; tdLNs, tumor-draining lymph nodes).

[0034] FIGURES 26A-26C depict representative tumor images from intravenous administration of NIRF nanosensors. FIGS. 26A-26B show fluorescent images of MC38 tumors (n = 5-7) from mice given intravenous administration of linear GzmB nanosensors (FIG.26A) or AND-gated nanosensors (FIG.26B) one day after the third ICB or Iso treatment dose. Images were taken 3 hours after sensor administration. FIG.26C shows quantified tumor NIRF (RFU / area; two-way ANOVA with Sidak’s post-test and correction for multiple comparisons).

[0035] FIGURE 27 depicts that AND-gated nanosensors increase tumor-to-organ selectivity compared to linear GzmB nanosensors. Tumor-to-organ selectivity is defined as the ratio of isotype-normalized fluorescence in ICB-treated tumors compared to the corresponding normalized fluorescence in a respective non-tumor organ (n = 6-7 biological replicates).

[0036] FIGURES 28A-28C depict that AND-gated nanosensors retain tumor-specific detection of ICB responses after chemotherapy. FIG. 28A shows a treatment schedule including a single dose of oxaliplatin (0.1 mg, i.p.), followed by three doses of either ICB or Iso. AND-gated nanosensors were administered i.v. one day after the third dose of ICB therapy. FIG. 28B shows tumor growth kinetics for mice treated with ICB and / or oxaliplatin (***P < 0.001, ****P < 0.0001, n = 4-7 biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIG. 28C shows representative fluorescent images of MC38 tumors and major organs harvested three hours after administration of AND-gated nanosensors.

[0037] FIGURES 29A-29B depict a treatment schedule and representative images for assessing specificity for colocalized proteases. FIGS. 29A-29B show a treatment schedule (FIG.29A) and representative fluorescent images (FIG.29B) for tumor-bearing mice infected with PR8 influenza A virus and treated with ICB therapy. Images are displayed at same scale as corresponding images of lungs and tumor in FIG.25E.

[0038] FIGURES 30A-30J depict that AND-gated nanosensors detect anti-tumour responses during ICBT. FIG. 30A shows tumour growth kinetics for C57BL / 6J (B6) mice bearing subcutaneous MC38 tumours. Starting on day 13, mice were treated every 3 days for up to 4 doses of immune checkpoint blockade (ICB) therapy or isotype (Iso) control treatment. ^Attorney Docket No.10034-321WO1 ICBT consisted of intravenous (i.v.) administration of 0.2 mg each of anti-PD1 and anti- CTLA4 antibodies and intraperitoneal administration of 10 ^g of IL-2 cytokine. Iso control treatment consisted of i.v. administration of 0.2 mg each of IgG1 and IgG2b isotype control antibodies (n = 8 (ICB) or 7 (Iso) biological replicates, two-way ANOVA with Sidak’s post- test and correction for multiple comparisons). FIGS.30B-30D show representative flow plots (FIG.30B), frequency (FIG.30C) and number per gram of tissue (FIG.30D) of GzmB+CD8+T cells isolated from MC38 tumours in mice treated with ICB therapy or Iso. Flow analysis was performed 1 day after the third treatment dose (n = 5 (Iso) or 7 (ICB) biological replicates, two-tailed Student’s t-test). FIGS. 30E-30G show a schematic (FIG. 30E), representative images (FIG.30F) and quantification (FIG.30G) of NIRF in MC38 tumours and major organs (liver, lungs, heart, spleen, kidneys, brain and tumour-draining lymph nodes (tdLNs)) upon intratumoral (i.t.) administration of quenched NIRF AND-gated nanosensors to tumour- bearing mice 1 day after the third ICB or Iso treatment dose. Images were taken 3 h after sensor administration. Relative fluorescence in each organ (RFU of organ / total RFU from all excised organs for each mouse) was normalized to the average signal for Iso-treated mice (n = 6 (Iso) or 7 (ICB) biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIGS.30H-30J show urinalysis schematic (FIG.30H), quantification (FIG. 30I) and receiver-operator characteristic (ROC) curves (FIG. 30J) of carboxyfluorescein (5(6)-FAM) reporters in urine of tumour-bearing mice receiving i.t. administration of 5(6)-FAM-labelled AND-gated nanosensors 1 day after the third treatment. Urine was collected 3 h after sensor administration. Reporters were purified from urine by immunoprecipitation and quantified by fluorescence, which was normalized to the average signal for Iso-treated mice at each timepoint. B2m– / –tumours were generated from MC38 cancer cells with knocked out expression of B2m (beta-2 microglobulin) (n = 6 (Iso), 8 (ICB) or 3 (ICB, B2m– / –tumour) biological replicates, two-way ANOVA with Tukey’s post-test and correction for multiple comparisons) (FIG.30C, ***P = 0.0005; FIG.30D, *P = 0.0349; FIG. 30G, ***P = 0.0009; FIG. 30I, *P = 0.0220, **P = 0.0045; FIGS. 30A-30J, NS, not significant; error bars depict mean ± s.e.m.; AUC, area under the ROC curve).

[0039] FIGURES 31A-31I depict that AND-gated nanosensors increase specificity by requiring co-localized proteases for activation. FIG.31A shows schematic and kinetics of body weight for the mouse model of viral infection using intranasal administration of PR8 influenza A (30 plaque-forming units) (n = 10 biological replicates, two-way ANOVA with Sidak’s post- test and correction for multiple comparisons). FIGS.31B-31C show representative flow plots (FIG.31B) and number (FIG.31C) of GzmB+CD8+T cells isolated from BALF and lungs of ^Attorney Docket No.10034-321WO1 naive and PR8-infected mice 8 days post-infection. Dump channel includes cells stained with anti-CD4, anti-CD19 and anti-NK1.1 antibodies (n = 3 biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIG.31D shows schematic of expected detection specificity for the AND-gated sensor. A linear GzmB sensor is expected to activate during both anti-viral responses to influenza infection and anti-tumour responses to ICB. By contrast, an AND-gated sensor is expected to be tumour specific and remain inactive during anti-viral responses. FIGS. 31E-31F show representative images (FIG. 31E) and quantification (FIG.31F) of NIRF (RFU) in lungs upon i.v. administration of quenched NIRF linear GzmB nanosensors or AND-gated nanosensors to PR8-infected mice 8 days post- infection. Images were taken 3 h after sensor administration (n = 5 biological replicates, two- way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIG. 31G shows a schematic of possible outcomes for AND-gated nanosensors in distal tumours after i.t. administration to a local tumour in a bilateral tumour model. If sensors were partially cleaved by GzmB in the local tumour, they would activate distally in both resistant B2m– / –and responsive wild-type (WT) tumours independent of GzmB expression in the distal tumour. By contrast, if sensors remained predominantly intact upon arrival to distal tumours, they would retain the ability to discriminate resistant and responsive tumours dependent on GzmB expression. FIGS.31H-31I show representative images of local and distal tumours (FIG.31H) and quantification of NIRF (RFU per area) in distal tumours (FIG. 31I) for bilateral tumours upon i.t. administration of quenched NIRF AND-gated nanosensors to local tumours (n = 8 (WT / WT and WT / B2m– / –) or 6 (B2m– / – / WT) biological replicates, one-way ANOVA with Dunnett’s post-test and correction for multiple comparisons) (FIG. 31A, **P = 0.0075; FIG. 31I, **P = 0.0032 (WT / WT versus WT / B2m– / –) or 0.0037 (WT / B2m– / –versus B2m– / – / WT); FIGS.31A-31I, ****P < 0.0001, NS, not significant; error bars depict mean ± s.e.m.).

[0040] FIGURES 32A-32F depict that AND-gated nanosensors increase selectivity of on- tumour detection. FIGS.32A-32B show a representative image (FIG.32A) and quantification (FIG.32B) of fluorescence in MC38 tumours and major organs produced by i.v. administration of NIRF AND-gated nanosensors to tumour-bearing mice 1 day after the third ICB or Iso treatment dose. Images were taken 3 h after sensor administration. Fluorescence (RFU per area) in each organ was normalized to the average signal for Iso-treated mice (n = 6 (Iso) or 7 (ICB) biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIG.32C shows average tumour-to- organ selectivity for each organ produced by linear GzmB nanosensors or AND-gated nanosensors. Tumour-to-organ selectivity is defined as the ratio of isotype-normalized fluorescence in ICB-treated tumours compared with ^Attorney Docket No.10034-321WO1 the corresponding normalized fluorescence in a respective non-tumour organ. Average selectivity was calculated using the mean from n = 6 (Iso) or 7 (ICB) biological replicates (two- tailed paired Student’s t-test). FIG. 32D shows quantification of fluorescence in MC38 tumours and major organs produced by i.v. administration of NIRF AND-gated nanosensors to tumour-bearing mice that were treated with one dose of oxaliplatin (0.1 mg, intraperitoneally) followed by three doses of ICB or Iso. Fluorescence (RFU per area) in each organ was normalized to the average signal for mice treated with Iso only (n = 4 (Iso), 6 (ICB) or 7 (Oxa + Iso, Oxa + ICB) biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons). FIGS. 32E-32F show quantification of fluorescence in MC38 tumours, lungs and other major organs (FIG.32E) and representative images (FIG.32F) after i.v. administration of NIRF AND-gated nanosensors to tumour-bearing mice infected with PR8 influenza A virus and treated with three doses of ICB or Iso. Corresponding images of the liver and spleen are in FIG.38B. Fluorescence (RFU per area) in each organ was normalized to the average signal for uninfected control mice treated with Iso only (n = 5 (PR8 + Iso), 6 (Iso only), 7 (PR8 + ICB, B2m– / –tumour) or 8 (PR8 + ICB) biological replicates, two-way ANOVA with Dunnett’s post-test and correction for multiple comparisons) (FIG.32B, *P = 0.0382, ***P = 0.0005; FIG. 32C, **P = 0.0073; FIG. 32D, **P = 0.0042; FIG. 32F, *P = 0.0377, **P = 0.0069; FIGS. 32A-32F, ****P < 0.0001; error bars depict mean ± s.e.m.; ndLNs, non- tumour-draining lymph nodes; tdLNs, tumour-draining lymph nodes).

[0041] FIGURES 33A-33B depict that AND-gated nanosensors differentiate ICB- responsive and resistant tumors. Representative images (FIG. 33A) and quantification (FIG. 33B) of NIRF (RFU / area) in wild-type (w.t.) andMC38 tumors upon i.t. administration of quenched NIRF AND-gated nanosensors to tumor-bearing mice one day after the third ICB or Iso treatment dose. Images were taken 3 hours after sensor administration. (*P = 0.0257 (w.t. / – versus w.t. / +) or 0.0255 (w.t. / + versus B2m– / – / +), n = 6 (B2m– / – / +), 7 (w.t. / –), or 8 (w.t. / +) biological replicates, one-way ANOVA with Dunnett’s post-test and correction for multiple comparisons; error bars depict mean ± s.e.m.)

[0042] FIGURES 34A-34B depict specificity of AND-gated nanosensors to GzmB activity in flu-infected lungs. Representative image and quantification of NIRF produced by i.v. administration of quenched fluorescent linear GzmB sensors (FIG. 34A) or AND-gated nanosensors (FIG. 34B) in major organs (kidneys, liver, lungs, spleen) of naïve or PR8- infected mice. Fluorescence (RFU) for each organ was normalized to the respective average signal in naïve mice. (FIGS. 34A-34B, n = 5 biological replicates, ****P < 0.0001, two-way ^Attorney Docket No.10034-321WO1 ANOVA with Sidak’s post-test and correction for multiple comparisons; ns, not significant; error bars depict mean ± s.e.m.).

[0043] FIGURES 35A-35B depict tissue fluorescence produced by intravenously administered linear GzmB nanosensors. Representative image (FIG. 35A) and quantification (FIG.35B) of fluorescence in MC38 tumors and major organs produced by i.v. administration of NIRF linear GzmB nanosensors to tumor-bearing mice one day after the third ICB or Iso treatment dose. Images were taken 3 hours after sensor administration. Fluorescence (RFU / area) in each organ was normalized to average signal for Iso-treated mice. (n = 5 (Iso) or 6 (ICB) biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons; ns, not significant; error bars depict mean ± s.e.m.).

[0044] FIGURES 36A-36C depict representative tumor images from intravenous administration of NIRF nanosensors. FIGS. 36A-36B show fluorescent images of MC38 tumors (n = 5 (linear, Iso), 6 (linear, ICB and AND, Iso), or 7 (AND, ICB) biological replicates) from mice given intravenous administration of linear GzmB nanosensors (FIG.36A) or AND- gated nanosensors (FIG. 36B) one day after the third ICB or Iso treatment dose. Images were taken 3 hours after sensor administration. FIG.36C shows quantified tumor NIRF (RFU / area; two-way ANOVA with Sidak’s post-test and correction for multiple comparisons; ***P = 0.0001, ****P <0.0001, error bars depict mean ± s.e.m.).

[0045] FIGURES 37A-37C depict that AND-gated nanosensors retain tumor-specific detection of ICB responses after chemotherapy. FIG. 37A shows a treatment schedule including a single dose of oxaliplatin (0.1 mg, i.p.), followed by three doses of either ICB or Iso. AND-gated nanosensors were administered i.v. one day after the third dose of ICB therapy. FIG. 37B shows tumor growth kinetics for mice treated with ICB and / or oxaliplatin (***P = 0.0003, ****P < 0.0001, n = 4 (Iso only), 6 (ICB only), or 7 (Iso + Oxa, ICB + Oxa) biological replicates, two-way ANOVA with Sidak’s post-test and correction for multiple comparisons; error bars depict mean ± s.e.m.). FIG.37C shows representative fluorescent images of MC38 tumors and major organs harvested three hours after administration of AND-gated nanosensors.

[0046] FIGURES 38A-38B depict treatment schedule and representative images for assessing specificity for colocalized proteases. Treatment schedule (FIG. 38A) and representative fluorescent images (FIG. 38B) for tumor-bearing mice infected with PR8 influenza A virus and treated with ICB therapy. Corresponding images of lungs and tumor are in Fig.32E.

[0047] FIGURE 39 depicts the source image for FIG.33A.

[0048] FIGURE 40 depicts the source image for FIG.34A. ^Attorney Docket No.10034-321WO1

[0049] FIGURE 41 depicts the source image for FIG.34B.

[0050] FIGURE 42 depicts the source image for FIG.35A.

[0051] FIGURE 43 depicts the source image for FIGS.36A-36B.

[0052] FIGURE 44 depicts the source image for FIG.37C.

[0053] FIGURE 45 depicts the source image for FIG.38B. DETAILED DESCRIPTION

[0054] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. DEFINITIONS

[0055] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0056] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0057] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0058] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the ^Attorney Docket No.10034-321WO1 other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0059] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0060] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub- ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0061] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some ^Attorney Docket No.10034-321WO1 circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0062] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.

[0063] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0064] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually ^Attorney Docket No.10034-321WO1 increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0065] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and / or pharmaceutical composition, by changing the disclosed compound and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0066] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

[0067] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0068] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0069] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof. ^Attorney Docket No.10034-321WO1

[0070] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0071] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0072] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0073] The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).

[0074] Reference also is made herein to peptides, polypeptides, proteins and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein ^Attorney Docket No.10034-321WO1 is defined as a polymer of amino acids, typically of length 100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110).

[0075] A “functional fragment” as referred to herein comprises a portion of a polypeptide which retains its functional ability.

[0076] As disclosed herein, exemplary peptides, polypeptides, proteins may comprise, consist essentially of, or consist of any reference amino acid sequence disclosed herein, or variants of the peptides, polypeptides, and proteins may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any amino acid sequence disclosed herein. Variant peptides, polypeptides, and proteins may include peptides, polypeptides, and proteins having one or more amino acid substitutions, deletions, additions and / or amino acid insertions relative to a reference peptide, polypeptide, or protein. Also disclosed are nucleic acid molecules that encode the disclosed peptides, polypeptides, and proteins (e.g., polynucleotides that encode any of the peptides, polypeptides, and proteins disclosed herein and variants thereof).

[0077] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, ^-alanine, ^-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2- Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2 -Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the ^Attorney Docket No.10034-321WO1 peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0078] The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C- terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0079] Variants comprising deletions relative to a reference amino acid sequence or nucleotide sequence are contemplated herein. A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation or both of a reference polypeptide or a 5 -terminal or 3 -terminal truncation or both of a reference polynucleotide).

[0080] Variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein. A “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference ^Attorney Docket No.10034-321WO1 sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide / amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and / or the C-terminal region of a polypeptide or the 5 -terminal region and / or the 3 terminal region of a polynucleotide. The term !at least a fragment" encompasses the full length polynucleotide or full length polypeptide.

[0081] Variants comprising insertions or additions relative to a reference sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.

[0082] Fusion proteins and fusion polynucleotides also are contemplated herein. A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof. The heterologous protein(s) may be fused at the N- terminus, the C-terminus, or both termini. A fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein.

[0083] A fusion polynucleotide refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3 end of a first polynucleotide to a 5 end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such ^Attorney Docket No.10034-321WO1 that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below).

[0084] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0085] A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0086] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

[0087] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0088] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 13, Cold Spring Harbor Press, Plainview N.Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid ^Attorney Docket No.10034-321WO1 may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0089] “Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

[0090] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated. PROTEASE ACTIVITY SENSORS

[0091] In some aspects, disclosed herein is a protease activity sensor including the sequence: X3-X1-k-R1-kpP-X4-k-X5-R2-k-X2-K-X6(SEQ ID NO: 1), wherein: k is D-lysine; p is D-proline; X1and X2are amino acids crosslinked by their side chains; X3is a quencher or an acetyl group and X4is a reporter molecule, or X3is a reporter molecule and X4is null or a quencher; X5is null or cysteine-D-lysine (Ck); X6is glycinamide; and R1and R2are protease cleavage sites for the same or different proteases.

[0092] In some aspects, X1is glutamate and X2is lysine; and X1and X2can be crosslinked by their side chains to form an amide moiety. In other aspects, X1is lysine and X2is glutamate; and X1and X2can be crosslinked by their side chains to form an amide moiety. In some aspects, X1is aspartate and X2is lysine; and X1and X2can be crosslinked by their side chains to form an amide moiety. In other aspects, X1is lysine and X2is aspartate; and X1and X2can be crosslinked by their side chains to form an amide moiety. ^Attorney Docket No.10034-321WO1

[0093] In some aspects, X1and X2are both cysteine; and X1and X2can be crosslinked by their side chains to form a disulfide bridge.

[0094] In some aspects, X3is a quencher and X4is a reporter molecule. In some aspects, X3is an acetyl group and X4is a reporter molecule. In some aspects, X3is a reporter molecule and X4is null. In some aspects, X3is a reporter molecule and X4is a quencher.

[0095] In some aspects, X5is null. In some aspects, X5is cysteine-D-lysine (Ck);

[0096] In some aspects, the protease activity sensor can have a sequence selected from:wherein X3is a quencher or an acetyl group and X4is a reporter molecule, or X3is a reporter molecule and X4is a quencher. In some aspects, X3is a quencher and X4is a reporter molecule. In some aspects, X3is an acetyl group and X4is a reporter molecule. In some aspects, X3is a reporter molecule and X4is a quencher.

[0097] In some aspects, the protease activity sensor can have a sequence selected from: X3-Ek-R1-kpPk-R2-kKK-X6(SEQ ID NO: 6), X3-Kk-R1-kpPk-R2-kEK-X6(SEQ ID NO: 7), X3-Ek-R1-kpPkCk-R2-kKK-X6(SEQ ID NO: 8), or X3-Kk-R1-kpPkCk-R2-kEK-X6(SEQ ID NO: 9), wherein X3is a reporter molecule.

[0098] In some aspects, the reporter molecule can be a fluorophore, an isotopically encoded peptide, or an amino acid linked to a fluorophore or isotopically encoded peptide by its side chain. For example, in some aspects, the reporter molecule can be luciferase (e.g., NanoLuc, GlowLuc, RedLuc, Emerald Luc, Luc2, Fluc, GLuc (Gaussia luciferase), CLuc (Cypridina luciferase), ELuc (Enhanced beetle luciferase), SLuc (Synthetic luciferase), pLuc (Photinus pyralis luciferase variant), RLuc (Renilla luciferase), ALuc (Artificial luciferase series), UltraLuc, TurboLuc), green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyane fluorescent protein (CFP), monomeric red fluorescent protein (mRFP), Discosoma striata (DsRed), mCherry, mOrange, tdTomato, mSTrawberry, mPlum, ^Attorney Docket No.10034-321WO1 photoactivatable GFP (PA-GFP), Venus, Kaede, monomeric kusabira orange (mKO), Dronpa, enhanced CFP (ECFP), Emerald, Cyan fluorescent protein for energy transfer (CyPet), super CFP (SCFP), Cerulean, photoswitchable CFP (PS-CFP2), photoactivatable RFP1 (PA-RFP1), photoactivatable mCherry (PA-mCherry), monomeric teal fluorescent protein (mTFP1), Eos fluorescent protein (EosFP), Dendra, TagBFP, TagRFP, enhanced YFP (EYFP), Topaz, Citrine, yellow fluorescent protein for energy transfer (YPet), super YFP (SYFP), enhanced GFP (EGFP), Superfolder GFP, T30 Sapphire, Fucci, mKO2, mOrange2, mApple, Sirius, Azurite, EBFP, EBFP2, 6-FAMTm, TETTm, JOETM HEXTM, VICO, cyanine 3, ROXTM, LC Red 640, cyanine 5, fluorescein isothiocyanate (FITC), rhodamine, TRITC (tetramethyl rhodamine isothiocyanate), Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, Texas Red, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, and Alexa Fluor 750, and / or an amino acid linked to any of the above fluorophores by its side chain.

[0099] In some aspects, when the reporter molecule is an amino acid linked to a fluorophore or isotopically encoded peptide by its side chain, the amino acid and the fluorophore or isotopically encoded peptide can be linked using click chemistry. For example, in some aspects, the amino acid side chain can include an alkyne group (e.g., L- proparglyglycine), and the fluorophore or isotopically encoded peptide can include an azide group. In other aspects, the amino acid side chain can include an azide group, and the fluorophore or isotopically encoded peptide can include an alkyne group.

[0100] In some aspects, the quencher can be Deep Dark Quenchers (Eurogentec), DABCYL, TAMRA, BHQ-1®, BHQ-2®, BHQ-3®, BBQC)-650, ECLIPSE, Iowa Black® quenchers, QSY, and / or an amino acid linked to any of the above quencher molecules by its side chain.

[0101] In some aspects, when the quencher is an amino acid linked to a quencher molecule by its side chain, the amino acid and the quencher molecule can be linked using click chemistry. For example, in some aspects, the amino acid side chain can include an alkyne group (e.g., L- proparglyglycine), and the quencher molecule can include an azide group. In other aspects, the amino acid side chain can include an azide group, and the quencher molecule can include an alkyne group.

[0102] In some aspects, R1and R2are protease cleavage sites for the same protease. In some aspects, R1and R2are protease cleavage sites for different proteases.

[0103] In some aspects, R1and / or R2can be cleavable by a protease that indicates presence of a disease or disorder. Proteases known to be associated with diseases or disorders include, ^Attorney Docket No.10034-321WO1 but are not limited to, serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin K, Cathepsin L, kallikreins, hK1, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-10 converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase, meprins, granzymes and dipeptidyl peptidase IV (DPPIV / CD26).

[0104] In some aspects, R1and / or R2can be cleavable by a cancer-associated protease or tumor-associated protease. Example cancer-associated protease or tumor-associated proteases include, but are not limited to, cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein 1, kallikrein 3 (PSA), kallikrein 10, kallikrein15, uPA, uPAR, caspases, matrix metalloproteinases such as MMP1, MMP2, MMP8, MMP9, MMP13, MMP14, and ADAM.

[0105] In some aspects, the cancer can be breast cancer, and R1and / or R2can include any one of SEQ ID NOS: 274-364. In some aspects, the cancer can be colon cancer, and R1and / or R2can include any one of SEQ ID NOS: 365-387. In some aspects, the cancer can be small cell lung carcinoma, and R1and / or R2can include any one of SEQ ID NOS: 388-407. In some aspects, the cancer can be ovarian cancer, and R1and / or R2can include any one of SEQ ID NOS: 398-417. In some aspects, the cancer can be prostate cancer, and R1and / or R2can include any one of SEQ ID NOS: 418-437. In some aspects, the cancer can be liver cancer, and R1and / or R2can include SEQ ID NO: 73, SEQ ID NO: 109, SEQ ID NO: 123, SEQ ID NO: 133, SEQ ID NO: 156, SEQ ID NO: 171, SEQ ID NO: 216, SEQ ID NO: 254, SEQ ID NO: 266, or any one of SEQ ID NOS: 438-448. In some aspects, the cancer can be adenocarcinoma, and R1and / or R2can include SEQ ID NO: 166, SEQ ID NO: 265, or any one of SEQ ID NOS: 449- 466. In some aspects, the cancer can be renal cancer, and R1and / or R2can include any one of SEQ ID NOS: 467-486.

[0106] In some aspects, R1and / or R2can be cleavable by a tissue-specific protease (e.g., a T-cell specific protease). Example tissue-specific proteases include, but are not limited to, neutrophil serine proteases such as cathepsin G, neutrophil elastase, and proteinase 3, mucosa- ^Attorney Docket No.10034-321WO1 associated lymphoid tissue 1 (MALT1), granzymes, and cysteine proteinases of the caspase family, such as caspase-3,-6,-7,-8.

[0107] In some aspects, the tissue can be kidney tissue, and R1and / or R2can include any one of SEQ ID NOS: 487-496. In some aspects, the tissue can be liver tissue, and R1and / or R2can include any one of SEQ ID NOS: 497-506. In some aspects, the tissue can be lung tissue, and R1and / or R2can include any one of SEQ ID NOS: 507-516. In some aspects, the tissue can be heart tissue, and R1and / or R2can include any one of SEQ ID NOS: 517-526. In some aspects, the tissue can be blood, and R1and / or R2can include any one of SEQ ID NOS: 527- 536. In some aspects, the tissue can be spleen tissue, and R1and / or R2can include any one of SEQ ID NOS: 537-546. In some aspects, the tissue can be lymph node tissue, and R1and / or R2can include any one of SEQ ID NOS: 547-556. In some aspects, the tissue can be stomach tissue, and R1and / or R2can include any one of SEQ ID NOS: 557-566. In some aspects, the tissue can be ovarian tissue, and R1and / or R2comprise any one of SEQ ID NOS: 567-576. In some aspects, the tissue can be uterine tissue, and R1and / or R2can include any one of SEQ ID NOS: 577-586. In some aspects, the tissue can be mammary gland tissue, and R1and / or R2can include any one of SEQ ID NOS: 587-596. In some aspects, the tissue can be prostate tissue, and R1and / or R2can include any one of SEQ ID NOS: 597-606. In some aspects, the tissue can be testicular tissue, and the prostate-cleavable linker can include any one of SEQ ID NOS: 607-616. In some aspects, the tissue can be intestinal tissue, and R1and / or R2can include any one of SEQ ID NOS: 617-626. In some aspects, the tissue can be bladder tissue, and R1and / or R2can include any one of SEQ ID NOS: 627-636. In some aspects, the tissue can be brain tissue, and R1and / or R2can include any one of SEQ ID NOS: 637-646. In some aspects, the tissue can be thymic tissue, and R1and / or R2can include any one of SEQ ID NOS: 647-656.

[0108] In some aspects, R1and / or R2can be cleavable by a protease expressed by an immune cell during an immune response to a disease or disorder. For example, in some aspects, R1and / or R2can be cleavable by a protease expressed by an immune cell during an anti-tumor response. Several proteases are known to be associated with immune responses and, in particular, anti-tumor responses, such as inflammation and programmed cell death (e.g., including apoptosis, pyroptosis and necroptosis). The activity levels of those proteases are accordingly indicative of immune system activity. Caspases (cysteine-aspartic proteases, cysteine aspartases or cysteine-dependent aspartate-directed proteases) are a family of protease enzymes including a cysteine in their active site that nucleophilically cleaves a target protein only after an aspartic acid residue. Caspase-1, Caspase-4, Caspase-5 and Caspase-11 are associated with inflammation. Serine proteases also function in apoptosis and inflammation ^Attorney Docket No.10034-321WO1 and their differential expression is therefore also indicative of an immune response. Immune cells express serine proteases such as granzymes, neutrophil elastase, cathepsin G, proteinase 3, chymase, and tryptase.

[0109] In some aspects, R1can be cleavable by a protease that indicates presence of a disease or disorder, and R2can be cleavable by a tissue-specific protease, or vice versa. Similarly, in some aspects, R1can be cleavable by a cancer-associated protease or tumor- associated protease, and R2can be cleavable by a tissue-specific protease, or vice versa This could, in some such aspects, limit off-target activation of the protease activity sensor in other organs.

[0110] In some aspects, R1can be cleavable by a protease that indicates presence of a disease or disorder, and R2can be cleavable by a protease expressed by an immune cell during an immune response to a disease or disorder, or vice versa. Similarly, in some aspect, R1can be cleavable by a cancer-associated protease or tumor-associated protease, and R2can be cleavable by a protease expressed by an immune cell during an anti-tumor response, or vice versa. This could, in some such aspects, provide confirmation that an immune response or an anti-tumor response is happening at the site of the disease or disorder or within the tumor, respectively.

[0111] In some aspects, R1and / or R2can be cleaved by CASP1 and can include any one of SEQ ID NOS: 25-34. In some aspects, R1and / or R2can be cleaved by CASP3 and can include any one of SEQ ID NOS: 35-44. In some aspects, R1and / or R2can be cleaved by CASP8 and can include any one of SEQ ID NOS: 45-54. In some aspects, R1and / or R2can be cleaved by CTSD and can include any one of SEQ ID NOS: 55-64. In some aspects, R1and / or R2can be cleaved by CTSE and can include any one of SEQ ID NOS: 65-74. In some aspects, R1and / or R2can be cleaved by CTSG and can include any one of SEQ ID NOS: 75-83. In some aspects, R1and / or R2can be cleaved by CTSS and can include any one of SEQ ID NOS: 84-93. In some aspects, R1and / or R2can be cleaved by thrombin and can include any one of SEQ ID NOS: 94-103. In some aspects, R1and / or R2can be cleaved by FAP and can include any one of SEQ ID NOS: 104-113. In some aspects, R1and / or R2can be cleaved by FXIA and can include any one of SEQ ID NOS: 114-123. In some aspects, R1and / or R2can be cleaved by GGT1 and can include any one of SEQ ID NOS: 124-133. In some aspects, R1and / or R2can be cleaved by GZMA and can include any one of SEQ ID NOS: 134-143. In some aspects, R1and / or R2can be cleaved by GZMB and can include any one of SEQ ID NOS: 144-153. In some aspects, R1and / or R2can be cleaved by H20S and can include any one of SEQ ID NOS: 154-163. In some aspects, R1and / or R2can be cleaved by KLK2 and can include any one of SEQ ID NOS: 164- ^Attorney Docket No.10034-321WO1 173. In some aspects, R1and / or R2can be cleaved by MMP1 and can include any one of SEQ ID NOS: 174-183. In some aspects, R1and / or R2can be cleaved by MMP10 and can include any one of SEQ ID NOS: 184-193. In some aspects, R1and / or R2can be cleaved by MMP12 and can include any one of SEQ ID NOS: 194-203. In some aspects, R1and / or R2can be cleaved by MMP13 and can include any one of SEQ ID NOS: 204-213. In some aspects, R1and / or R2can be cleaved by MMP2 and can include any one of SEQ ID NOS: 214-223. In some aspects, R1and / or R2can be cleaved by MMP3 and can include any one of SEQ ID NOS: 224-233. In some aspects, R1and / or R2can be cleaved by MMP8 and can include any one of SEQ ID NOS: 234-243. In some aspects, R1and / or R2can be cleaved by MMP9 and can include any one of SEQ ID NOS: 244-253. In some aspects, R1and / or R2can be cleaved by TPA and can include any one of SEQ ID NOS: 254-263. In some aspects, R1and / or R2can be cleaved by UPA and can include any one of SEQ ID NOS: 264-273.

[0112] In some aspects, R1and / or R2are each independently selected from IEFDSG (SEQ ID NO: 10), APAALRAA (SEQ ID NO: 11), AAN (SEQ ID NO: 12), ASGPAGPA (SEQ ID NO: 13), PAALRA (SEQ ID NO: 14), fPRSG (SEQ ID NO: 15), LVXXXSG (SEQ ID NO: 19), LVSPRSG (SEQ ID NO: 20), LVSFPSG (SEQ ID NO: 21), LVQNLSG (SEQ ID NO: 22), LVPRGSG (SEQ ID NO: 23), and PLGLAG (SEQ ID NO: 24).

[0113] In some aspects, R1and R2can be different sequences that are cleaved by the same protease. In other aspects, R1and R2can have the same sequence. NANOPARTICLES

[0114] In some aspects, disclosed herein is a nanoparticle including a plurality of any of the disclosed protease activity sensors anchored to a surface of said nanoparticle. The nanoparticle can be any suitable nanoparticle known in the art, for example, a metal nanoparticle (e.g., iron oxide nanoparticle, gold nanoparticle, etc.), a polymeric nanoparticle (e.g., a PEG-based nanoparticle), a lipid nanoparticle, or any other suitable nanoparticle.

[0115] In some aspects, the nanoparticle can include a plurality of thiol-reactive moieties on its surface; X5is Ck; and the thiol-reactive moieties can be crosslinked to the cysteine in X5, thereby anchoring the plurality of protease activity sensors to the surface of the nanoparticle. For example, in some aspects, the plurality of thiol-reactive moieties can include iodoacetyl, maleimide, or any combination thereof.

[0116] In some aspects, the nanoparticle can include about 2 or more protease activity sensors anchored on its surface (e.g., about 3 or more, about 4 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 60 or more, about 70 or ^Attorney Docket No.10034-321WO1 more, about 80 or more, about 90 or more, about 100 or more, about 120 or more, about 140 or more, about 160 or more, about 180 or more, about 200 or more). In some aspects, the nanoparticle can include about 200 or less protease activity sensors anchored on its surface (e.g., about 180 or less, about 160 or less, about 140 or less, about 120 or less, about 100 or less, about 90 or less, about 80 or less, about 70 or less, about 60 or less, about 50 or less, about 45 or less, about 40 or less, about 35 or less, about 30 or less, about 25 or less, about 20 or less, about 15 or less, about 10 or less, about 5 or less, about 4 or less, about 3 or less, about 2 or less).

[0117] The nanoparticle can include any number of protease activity sensors anchored on its surface ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 2 to about 200 protease activity sensors anchored on its surface (e.g., from about 3 to about 180, from about 4 to about 160, from about 5 to about 140, from about 10 to about 120, from about 15 to about 100, from about 20 to about 90, from about 25 to about 80, from about 30 to about 70, from about 35 to about 60, from about 40 to about 50, from about 2 to about 45, from about 3 to about 40, from about 4 to about 35, from about 5 to about 30, from about 10 to about 25, from about 15 to about 20, from about 45 to about 200, from about 50 to about 180, from about 60 to about 160, from about 70 to about 140, from about 80 to about 120, from about 90 to about 100).

[0118] In some aspects, upon cleavage of both R1and R2, the reporter molecule can remain anchored to the surface of the nanoparticle. In some such aspects, X3is a quencher and X4is the reporter molecule. In other such aspects, X3is an acetyl group and X4is the reporter molecule.

[0119] In some aspects, upon cleavage of both R1and R2, the reporter molecule can be released from the nanoparticle. In some such aspects, X3is the reporter molecule and X4is null. In other such aspects, X3is the reporter molecule and X4is a quencher. METHODS

[0120] In some aspects, disclosed herein is a method of detecting presence of a protease, the method including: a) providing any of the disclosed protease activity sensors or any of the disclosed nanoparticle to a sample or a subject, wherein R1and R2are protease cleavage sites for the same protease; and b) detecting presence or absence of the reporter molecule in the sample, the subject, or a sample taken from the subject, thereby detecting presence or absence of the protease; wherein presence of the reporter molecule indicates presence of the protease; and wherein absence of the reporter molecule indicates absence of the protease. ^Attorney Docket No.10034-321WO1

[0121] In some aspects, step a) can further include: i) providing the protease activity sensor to the subject; and ii) collecting a biological fluid sample (including, but not limited to urine, blood, saliva, breath, sputum, bone marrow, cerebrospinal fluid (CSF), plasma, synovial fluid, nasal lavage, cell lysate, oral mucosa, nasal mucosa, vaginal mucosa, and / or rectal mucosa) from the subject; and step b) can further include detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the protease.

[0122] In some aspects, step a) can further include providing the nanoparticle to the subject, wherein upon cleavage of both R1and R2, the reporter molecule remains anchored to the surface of the nanoparticle; and step b) can further include imaging the subject to detect presence or absence of the reporter molecule, thereby locally detecting presence or absence of the protease.

[0123] In some aspects, step a) can further include: i) providing the nanoparticle to the subject, wherein, upon cleavage of both R1and R2, the reporter molecule is released from the nanoparticle; and ii) collecting a biological fluid sample (including, but not limited to urine, blood, saliva, breath, sputum, bone marrow, cerebrospinal fluid (CSF), plasma, synovial fluid, nasal lavage, cell lysate, oral mucosa, nasal mucosa, vaginal mucosa, and / or rectal mucosa) from the subject; and step b) can further include detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the protease.

[0124] In some aspects, presence of protease can indicate presence and / or severity of a disease or disorder; and the method can be used to determine if the subject has said disease or disorder and / or progression of said disease or disorder.

[0125] In some aspects, presence of the protease can indicate an immune response to a disease or disorder; and the method can be used to determine responsiveness of said disease or disorder to a treatment. For example, in some aspects, the treatment can be an immunotherapy, for example, an immune cell therapy (such as, for example, T cell therapy, macrophage therapy, NK cell therapy, NK T cell therapy, CAR T cell therapy, and / or CAR NK cell therapy) and / or one or more checkpoint inhibitors) and / or administration of one or more checkpoint inhibitors. Examples of checkpoint inhibitors include, but are not limited to, a PD-L1 inhibitor, a PD-1 inhibitor, and a CTLA-4 inhibitor. In some embodiments, the PD-L1 inhibitor includes, but is not limited to Atezolizumab, Avelumab, Durvalumab, LY3300054 (Eli Lilly and Company), and monoclonal antibodies or monoclonal antibody conjugates that act as a PD-L1 inhibitor. In some embodiments, the PD-1 inhibitor includes, but is not limited to pembrolizumab, Nivolumab, Cemiplimab and monoclonal antibodies or monoclonal antibody conjugates that ^Attorney Docket No.10034-321WO1 act as a PD-1 inhibitors. In some embodiments, the CTLA-4 inhibitor includes, but is not limited to Ipilimumab, AGEN1884 and monoclonal antibodies or monoclonal antibody conjugates that act as a CTLA-4 inhibitor.

[0126] In some aspects, the disease or disorder can be cancer. Example cancers include, but are not limited to, breast cancer, lung cancer, pancreatic cancer, gastric cancer, colon cancer, acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B- cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T- ^Attorney Docket No.10034-321WO1 lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T- cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T- cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's disease of the vulva).

[0127] In some aspects, the subject can have a tumor; and step a) can further include injecting the protease activity sensor or the nanoparticle into said tumor.

[0128] In some aspects, also disclosed herein is a method of detecting presence of two proteases, the method including: a) providing any of the disclosed protease activity sensors or ^Attorney Docket No.10034-321WO1 any of the disclosed nanoparticles to a sample or a subject, wherein R1and R2are protease cleavage sites for different proteases; and b) detecting presence or absence of the reporter molecule in the sample, the subject, or a sample taken from the subject, thereby detecting presence or absence of the two proteases; wherein presence of the reporter molecule indicates presence of both of the two proteases; and wherein absence of the reporter molecule indicates absence of one or both of the two proteases.

[0129] In some aspects, step a) can further include: i) providing the protease activity sensor to the subject; and ii) collecting a biological fluid sample (including, but not limited to urine, blood, saliva, breath, sputum, bone marrow, cerebrospinal fluid (CSF), plasma, synovial fluid, nasal lavage, cell lysate, oral mucosa, nasal mucosa, vaginal mucosa, and / or rectal mucosa) from the subject; and step b) can further include detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the two proteases.

[0130] In some aspects, step a) can further include providing the nanoparticle to the subject, wherein upon cleavage of both R1and R2, the reporter molecule remains anchored to the surface of the nanoparticle; and step b) can further include imaging the subject to detect presence or absence of the reporter molecule, thereby locally detecting presence or absence of the two proteases.

[0131] In some aspects, step a) can further include: i) providing the nanoparticle to the subject, wherein, upon cleavage of both R1and R2, the reporter molecule is released from the nanoparticle; and ii) collecting a biological fluid sample (including, but not limited to urine, blood, saliva, breath, sputum, bone marrow, cerebrospinal fluid (CSF), plasma, synovial fluid, nasal lavage, cell lysate, oral mucosa, nasal mucosa, vaginal mucosa, and / or rectal mucosa) from the subject; and step b) can further include detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the two proteases.

[0132] In some aspects, presence of both of the two proteases can indicate presence and / or severity of a disease or disorder; and the method can be used to determine if the subject has said disease or disorder and / or progression of said disease or disorder.

[0133] In some aspects, presence of both of the two protease can indicate an immune response to a disease or disorder; and the method can be used to determine responsiveness of said disease or disorder to a treatment. For example, in some aspects, the treatment can be an immunotherapy, for example, an immune cell therapy (such as, for example, T cell therapy, macrophage therapy, NK cell therapy, NK T cell therapy, CAR T cell therapy, and / or CAR NK ^Attorney Docket No.10034-321WO1 cell therapy) and / or one or more checkpoint inhibitors) and / or administration of one or more checkpoint inhibitors. Examples of checkpoint inhibitors are described above.

[0134] In some aspects, the disease or disorder can be cancer. Examples of cancers are described above.

[0135] In some aspects, the subject can have a tumor; and step a) can further include injecting the protease activity sensor or the nanoparticle into said tumor.

[0136] In some aspects, any of the disclosed protease activatable receptors or nanoparticles can be administered periodically, for example, to track progression of a disease or disorder over time. For example, in some aspects, any of the disclosed protease activatable receptors or nanoparticles can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more times. In some aspects, any of the disclosed protease activatable receptors or nanoparticles can be administered daily. In some aspects, any of the disclosed synthetic nucleic acid sequences can be administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or more. In some aspects, any of the disclosed synthetic nucleic acid sequences can be administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In some aspects, any of the disclosed protease activatable receptors or nanoparticles can be administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more. In some aspects, any of the disclosed protease activatable receptors or nanoparticles can be administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more. EXAMPLES Example 1: Monitoring immune dynamics in the tumor microenvironment via AND- gated protease sensors

[0137] Immune checkpoint blockade (ICB) therapy has transformed the treatment of cancer for patients across a broad range of malignancies, yet response rates remain low for the majority of cancer types. The outcome of ICB therapy largely depends on factors that vary across patients and tumor types, such as mutational burden, PD-L1 expression, resistance mutations, and immune cell infiltration (“hot” and “cold” tumors). The vast majority of these factors are assessed by biopsies, which are invasive and provide static information. Therefore, there is a need for strategies to assess dynamic changes in the tumor microenvironment to ^Attorney Docket No.10034-321WO1 identify biomarkers of response. One opportunity is the central role of proteases in antitumor immunity. Immune cells that predominate in hot tumors, such as CD8+ T cells and M1 macrophages, are associated with differential protease expression compared to suppressive cells in cold tumors like M2 macrophages and cancer-associated fibroblasts. For instance, granzyme B activity by CD8+ T cells is an established hallmark of responses to ICB therapy that is being leveraged to design imaging probes and synthetic biomarkers. However, immune cells are also active outside the tumor, especially in the context of ICB therapy that has high incidence of immune-related adverse events, and next-generation diagnostics should specifically report on-tumor activity. Leveraging the dysregulation of matrix metalloproteinases (MMPs) across cancer types, a study was conducted which designed AND- gated protease sensors that are activated if and only if they are cleaved by both MMPs and granzyme B, thereby sensing T cell activity selectively in the tumor to indicate responses to ICB therapy. Materials and Methods

[0138] The study synthesized AND-gated sensors including cyclic peptides, with substrates for both GzmB and MMPs and a fluorescent reporter, which were crosslinked to iron oxide nanoparticles. Cleavage activity was assessed in vitro by monitoring fluorescence in the presence of recombinant GzmB and / or MMPs. The study then assessed activation during anticancer T cell killing by culturing activated OT1 T cells with OVA-pulsed MC38 cancer cells, then incubating AND-gated sensors with conditioned media. For animal models, 106 MC38 cancer cells were subcutaneously inoculated in B6 mice. After tumors reached ~400 mm3, mice were treated with a combination of ^PD1, ^CTLA4, and IL2, or control antibodies. To assess sensor activation, AND-gated sensors bearing near-infrared fluorophore were administered intratumorally one day after the third dose, then tumor and organs were isolated after 3 hours and fluorescence was quantified by near-infrared imaging. Results and Discussion

[0139] The AND-gated sensors are actuated by cyclic peptides bearing substrates for two different proteases such that both substrates must be cleaved to produce a fluorescent signal. Using recombinant proteases, it was confirmed that AND-gated sensors were not activated in the presence of a single protease and were activated only when both GzmB and an MMP were present. The study then tested if AND-gated sensors are sensitive to cell-secreted proteases during T cell killing of cancer cells. OVA-specific OT1 T cells were cultured with MC38 cancer cells pulsed with OVA antigen. AND-gated sensors produced elevated fluorescence only in the context of T cells and OVA-pulsed cancer cells, indicating they are activated by ^Attorney Docket No.10034-321WO1 and selective for anticancer T cell cytotoxicity. Finally, the study confirmed activation in antitumor responses in a mouse model of ICB therapy. Mice bearing MC38 tumors were treated with combination ICB, which led to reduced tumor burden. After three doses, AND-gated sensors intratumorally injected, then tumor and organs were isolated to quantify the distribution of activated sensors. A significant 7-fold increase in the relative signal was observed in the tumor during ICB treatment, indicating that AND-gated sensors report on T cell activity during ICB responses. Conclusions

[0140] The ability to monitor the activity of immune cells selectively in the tumor microenvironment enables assessment of hot and cold immune infiltrates to predict responses pre- and early-on-treatment. Results demonstrate that intratumoral administration of AND- gated sensors sensitive to granzyme B and MMPs leads to tumor-selective activation during ICB responses. Example 2: Activity-based nanosensors that implement AND-gate logic for programmable detection of antitumor immunity

[0141] While the gene circuit analogy will remain at the forefront of development, advancing the design of cell- and gene-free biocircuits is gaining increased attention based on their potential to lower barriers for clinical translation while retaining the ability to perform logical functions. For example, RNA-based switches that can regulate protein translation in response to cell-type or cell-state specific biomarkers or externally applied small molecule inducers can be delivered to cells via lipid nanoparticles without the need for genetic engineering of cells. Similarly, protein circuits that do not primarily rely on transcriptional regulation have been engineered as logic gates, bandpass filters, and regulatory cascades for antigen sensing, protein secretion, and activation of enzymes and transcription factors. Recent work has also described nanomaterials comprised entirely of synthetic components that can interface with living cells and carry out computations to process information from biological inputs, often in the form of cell-secreted proteases. These include the development of peptide- caged liposomes as bio-comparators for analog-to-digital conversion of protease activity and hairpin peptide prodrugs that require proteolytic processing to digitize drug delivery. Optical imaging probes have similarly used protease activity to improve contrast and specificity, including ‘hub and spoke’ fluorogenic peptides that require proteolytic release of multiple quenchers to highlight tumor margins and tandem-activatable molecular probes that fluoresce after sequential cleavage by an endoprotease followed by a tumor-associated exoprotease. Such activity-based approaches can also take advantage of the promiscuous activity of proteases in ^Attorney Docket No.10034-321WO1 an analog computing framework, rather than one in binary, to solve mathematical problems and classify biological samples. These examples highlight the growing potential of computational nanomaterials that, despite being cell- and gene-free, can apply logic as a means to increase detection precision.

[0142] Here, a study was conducted which describes the development of a cell- and gene- free design of AND-gated nanosensors that require a specific pair of proteases before sensor activation and the release of a reporter that can be detected locally in tissue or distally in the urine. AND- gated nanosensors are implemented using asymmetric bi-labile cyclic peptides that actuate by releasing a reporter if and only if both flanking substrates are cleaved. The cyclic peptides are multivalently displayed on an iron oxide nanoparticle (IONP) to improve the catalytic efficiency of proteolysis and tissue retention in vivo. In mouse models, the study demonstrates that an AND-gated nanosensor designed to activate by a combination of matrix metalloproteinases (MMPs) expressed in tumors and granzyme B (GzmB) secreted by cytotoxic T cells can selectively report on antitumor responses during immune checkpoint blockade (ICB) therapy while minimizing activation from off-tumor sites such as in the lung during an acute viral infection. This study outlines a general approach to design AND-gated nanosensors that implement logic to improve the specificity of detecting disease and monitoring drug response by requiring the activity of paired proteases as inputs. Methods

[0143] Animals: Female mice (6- to 10-week-old) were used at the outset of all experiments. OT-1 (C57BL / 6-Tg(TcraTcrb)1100Mjb / J) transgenic mice were bred in-house using breeding pairs purchased from The Jackson Laboratory (Jax, 003831). C57BL / 6J (B6) mice were purchased from Jax (000664). All animal procedures were approved by the Georgia Tech Institutional Animal Care and Use Committee (protocol no. KWONG-A100190, KWONG-A100191, and KWONG-A100193).

[0144] Peptide synthesis: Peptides were either synthesized in house or ordered from a vendor (Lifetein, Genscript, or CPC Scientific). In-house synthesis was performed by Fmoc solid phase peptide synthesis on a Liberty Blue peptide synthesizer (CEM) using low-loading rink amide resin (CEM). Briefly, for each amino acid cycle, Fmoc deprotection was performed using 20% (v / v) 4-methylpiperidine in dimethylformamide, and amino acids (5 equivalents (eq.); Chem Impex) were coupled in the presence of 10 eq. diisopropylcarbodiimide (DIC) and 5 eq. Oxyma Pure. After synthesis, peptides were cleaved off resin using 92.5% trifluoroacetic acid (TFA), 2.5% water, 2.5% triisopropylsilane, and 2.5% 3,6-dioxa-1,8-octane-dithiol for 30- 45 minutes at 41C using Razor (CEM). After cleavage, peptides were precipitated in ice-cold ^Attorney Docket No.10034-321WO1 diethyl ether and vacuum dried overnight. Crude peptides were purified by reverse phase high- performance liquid chromatography (HPLC; 1260 Infinity II, Agilent) using a Zorbax C18 column (Agilent) using an elution gradient from 5% to 100% acetonitrile in 0.05% TFA. Acetonitrile was removed from purified fractions by rotary evaporation, and samples were frozen overnight and lyophilized.

[0145] To make cyclic peptides, linear peptides were first synthesized with Lys(Alloc) near the C-terminus and Glu(OAllyl) at the N-terminus. Fmoc-protected resin was then treated with phenylsilane and tetrakis(triphenylphosphine)palladium(0) in dichloromethane at 35 °C to restore Lys and Glu side chains, followed by cyclization using DIC and Oxyma.

[0146] For labeling with fluorophores and quenchers, resin-coupled peptides were N- terminally labeled with 5(6)-carboxyfluorescein (FAM) directly on Liberty Blue with DIC and Oxyma pure after Fmoc deprotection. Alternatively, Fmoc deprotection was performed on Liberty Blue, and resin-coupled peptides were N-terminally labeled with Tide Quencher 7WS (TQ7) succinimidyl ester (AAT Bioquest) in dimethyl sulfoxide with 4-10 eq. triethylamine at 37C for 2 hours. Peptides, which were synthesized with propargylglycine, were cleaved from resin and labeled with Tide Quencher 2 (TQ2) azide (FAM peptides; AAT Bioquest) or sulfo- cyanine7 azide (Lumiprobe) by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). CuAAC reaction was performed using copper(II) sulfate, tris(3- hydroxypropyltriazolylmethyl)amine (THPTA), ascorbic acid, and aminoguanidine in phosphate-buffered saline at 37C overnight. Peptides were purified by HPLC after dye labeling.

[0147] Mass spectrometric analysis of synthetic peptides and cleavage products: Mass analysis of crude and purified peptides synthesized in house was performed by liquid chromatography-mass spectrometry (LC / MS; 1260 Infinity II and InfinityLab LC / MSD, Agilent; OpenLab ChemStation software) on positive ion mode electrospray ionization (ESI / MS) using a Zorbax or Poroshell C18 column (Agilent) and an elution gradient from 5% to 100% acetonitrile in either 0.05% TFA or 0.05% formic acid (FA). Mass analysis of peptide cleavage products was performed by ESI / MS (Agilent) or by matrix-assisted laser desorption / ionization mass spectrometry (MALDI / MS; Autoflex, Bruker) using an ^-cyano-4- hydroxycinnamic acid matrix. Prior to analysis, cleavage products were diluted to 75% acetonitrile and refrigerated for 30 minutes to precipitate proteins, followed by centrifugation at 4000xg for 5 minutes.

[0148] Nanosensor conjugation: Amine-functionalized iron oxide nanoparticles (IONPs) were synthesized in-house as previously described and purified by fast-performance liquid ^Attorney Docket No.10034-321WO1 chromatography (FPLC; AKTA Pure, GE Healthcare; Unicorn software) using a Superdex 200 Increase 10-300 GL column, resulting in particles of ~20 nm average hydrodynamic diameter as determined by dynamic light scattering (Zetasizer, Malvern). For conjugation to peptides, nanoparticles were labeled with SM(PEG)6 (ThermoFisher) for 2 hours, then crosslinked to peptides containing free cysteines overnight. Reactions were performed in phosphate-buffered saline (PBS; pH 7.2-7.4) at room temperature; 2 mM ethylenediaminetetraacetic acid was added to the peptide crosslinking reaction to reduce formation of disulfide bonds between peptides. Nanosensors were purified by FPLC. Nanosensor valency (i.e., peptide-to- nanoparticle ratio) was quantified by UV / Vis spectroscopy, using 400 nm for IONPs and 490 nm for FAM / TQ2-labeled peptides or 750 nm for Cy7 / TQ7-labeled peptides. All valency calculations were performed using optical density (OD) values below 2.0 to ensure linearity of the Beer-Lambert Law. Samples were diluted in PBS to achieve OD < 2.0 when necessary.

[0149] Cell culture: MC38 cells (kind gift of the National Cancer Institute and Dr. Dario Vignali, University of Pittsburgh) and B2m− / −MC38 cells (developed in previous work) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin– streptomycin (PenStrep; ThermoFisher). Cells were grown to 70-90% confluence before being trypsinized and passaged with a split ratio of 20-40:1. For OT-1 T cells, OT-1 transgenic mice were sacrificed, primary splenocytes were harvested, and CD8 T cells were isolated (CD8a+ T cell isolation kit, Miltenyi). T cells were activated for 48 hours using ^CD3e and ^CD28 antibodies (BD Biosciences; clones 145-2C11 and 37.51, respectively) and cultured for up to 4 days at <3 million cells per mL, using T cell media (RPMI 1640 supplemented with 10% FBS, 1% PenStrep, 1% non-essential amino acids, 1% sodium pyruvate, 4 ppm (v / v) beta- mercaptoethanol, and 30 units / mL IL-2 (Roche)).

[0150] Fluorogenic substrate assay: Granzyme B (GzmB; Peprotech 140-03), thrombin (Thrb; Prolytix HCT-0020), fibroblast activation protein (FAP; R&D Systems 3715-SE), gamma-glutamyltransferase 1 (GGT1; R&D Systems 10977-GT), granzyme A (GZMA; Enzo Life Sciences ALX-201-118), ADAMTS1 (R&D Systems 2197-AD), tissue-type plasminogen activator (TPA; R&D Systems 7449-SE), urokinase-type plasminogen activator (UPA; R&D Systems 1310-SE), and plasmin (PLG; Prolytix HCPM-0140) were diluted directly in PBS. Matrix metalloproteinases (MMPs; Enzo Life Sciences BML-AK013 and BML-AK014) were diluted in MMP activation buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.1 mM ZnCl2, 0.05% Brij-35, pH 7.4) and activated for 30 minutes at 37 °C prior to dilution in PBS. Legumain (LGN; R&D Systems 2058-CY) and cathepsins (CTSD, CTSE, CTSS; Enzo Life Sciences BML-SE199, R&D Systems 1294-AS, R&D Systems 1183-CY) were diluted in LGN ^Attorney Docket No.10034-321WO1 activation buffer (100 mM NaOAc, 100 mM NaCl, pH 4.5; 20 mM DTT was added for CTSS) and activated at 37 °C prior to dilution in PBS to final pH ~5.5. Caspases (CASP1, CASP3, CASP8; Enzo Life Sciences BML-SE168, BML-SE169, BML-SE172) were diluted in caspase activation buffer (50 mM HEPES, 100 mM NaCl, 20 mM DTT, 2 mM EDTA, 0.1% Tween- 20, 10% glycerol) and activated for 30 minutes at 37 °C prior to dilution in PBS. Conditioned media was isolated freshly from cultured cells by centrifugation at 1000xg for 5 minutes. Peptides or nanosensors were mixed with recombinant proteases or conditioned media and incubated at 37 °C. For peptides labeled with FAM and TQ2, sample fluorescence was measured by Cytation 5 plate reader (Biotek; Gen5 software). For peptides labeled with Cy7 and TQ7, sample fluorescence was measured by Odyssey CLx Imager (LI-COR; ImageStudio software). Peptide concentrations were 1-10 ^M for free peptides in solution and 0.1-1 µM for nanosensors. Protease concentrations were 50-250 nM unless otherwise specified.

[0151] T cell cytotoxicity assay: Five days after harvesting OT-1 T cells, MC38 cells were trypsinized and incubated at 1 million cells per mL with 30 ^M antigen peptide (OVA or gp34) in OptiPro for 1 hour at 37 °C. Antigen-pulsed MC38 cells were washed in T cell media, and 60,000 cells were seeded in a 96-well plate. After 4-6 hours, OT-1 T cells were washed in T cell media, and 300,000 cells were added to the tumor cells (effector:target ratio = 5:1). After overnight incubation (12-16 hours), conditioned media was isolated by centrifugation. Conditioned media was either used for fluorogenic substrate assay or analyzed by lactate dehydrogenase assay (Abcam) or enzyme-linked immunosorbent assay for GzmB (ThermoFisher) or MMP9 (Abcam).

[0152] Mouse models of cancer immune checkpoint blockade therapy: B6 mice were subcutaneously inoculated into the left flank with 1 million MC38 cells or B2m− / −MC38 cells. Both left and right flanks were inoculated for the bilateral tumor model. Tumor burden (0.52^×^length^×^width^×^depth) was monitored twice weekly, and treatment was initiated at 300-400^mm3. 0.2 milligrams each of ^PD1 antibody (kind gift of Dr. Gordon Freeman of Dana-Farber, with the help of Dr. Rafi Ahmed of Emory University, clone 8H3) and ^CTLA4 antibody (BioXCell; clone 9H10) or matched IgG1 and IgG2b isotype controls (BioXCell; clones MOPC-21 and MPC-11, respectively) were intravenously administered by tail vein injection every 3 days for up to 4 doses. 10 ^g of IL-2 (Peprotech) was intraperitoneally administered to mice receiving ^PD1 / ^CTLA4 treatment on the same schedule. For chemotherapy, 0.1 mg of oxaliplatin (Thermo Fisher J66586.MF) was intraperitoneally administered 3 days before initiation of ICB therapy. For tumor-bearing mice infected with PR8 influenza A, mice were infected as described below one day before initiation of ICB ^Attorney Docket No.10034-321WO1 therapy. Flow cytometric analysis and intratumoral or intravenous injection of nanosensors for near-infrared fluorescence (NIRF) imaging or urinalysis were performed one day after the third treatment dose.

[0153] Mouse models of influenza A virus: B6 mice were intranasally inoculated with 30 plaque-forming units (p.f.u.) of purified H1N1 influenza A virus (PR8; strain A / PR / 8 / 34). Body weight was measured daily. Flow cytometric analysis and intravenous injection of nanosensors for near-infrared fluorescence (NIRF) imaging were performed eight days post infection.

[0154] Organ dissociation and flow cytometry analysis: For tumor-bearing mice, MC38 tumors were enzymatically and mechanically dissociated using mouse tumor dissociation kit (Miltenyi) and gentleMACS dissociator (Miltenyi), respectively. Tumor-infiltrating lymphocytes (TILs) were isolated from the single-cell suspension using a density gradient with Percoll centrifugation media (GE Life Sciences) and RPMI 1640 at 44:56 volume ratio, followed by red blood cell (RBC) lysis. For PR8-infected mice and naïve controls, bronchoalveolar lavage fluid (BALF) was collected using 3 washes with 1 mL of PBS. Cells were harvested from BALF by centrifugation, followed by RBC lysis. Lungs were enzymatically dissociated using collagenase type IV. Lymphocytes were isolated from the single-cell suspension using a 40:60 Percoll:RPMI density gradient, followed by RBC lysis.

[0155] For flow cytometry analysis, cells were first stained for surface markers in FACS buffer (1x DPBS, 2% FBS, 1^mM EDTA, 25^mM HEPES), followed by viability staining with LIVE / DEAD Fixable Near-IR dye (Invitrogen). Cells were fixed and permeabilized (eBioscience) prior to intracellular staining for GzmB in permeabilization buffer. All antibodies were used for staining at 1:100 dilution from stock concentrations. Stained cells were analyzed using an LSRFortessa (BD Biosciences; FACSDiva software) or Aurora (Cytek Biosciences; SpectroFlo software) flow cytometer. To determine absolute cell counts, CountBright Absolute Counting Beads (ThermoFisher) were added to samples immediately prior to analysis.

[0156] Antibody clones used were ^CD45 (30-F11), ^CD3 (145-2C11), ^CD8 (53-6.7), ^CD4 (RM4-5), ^NK1.1 (PK136), ^CD19 (6D5), and ^GZMB (GB12). ^GZMB was purchased from ThermoFisher; all other antibodies were purchased from Biolegend.

[0157] Near-infrared fluorescent imaging of sensor activation in whole organs and homogenates: Three hours after injection of NIRF Cy7 / TQ7-labeled nanosensors, tumor- bearing or PR8-infected mice were euthanized, and tumor and organs (brain, lungs, heart, liver, kidneys, spleen, tumor-draining and non-tumor-draining lymph nodes) were isolated, rinsed in ^Attorney Docket No.10034-321WO1 PBS, and imaged by Odyssey CLx (LI-COR; ImageStudio software). Alternatively, tumor and organs were isolated from mice that did not receive NIRF nanosensors and were dissociated using FastPrep-24 homogenizer (MP Biomedicals) with beads (Lysing Matrix D, MP Biomedicals) in tissue protein extraction buffer (T-PER, ThermoFisher). Homogenate was isolated by centrifugation at 14,000xg for 5 minutes and added to GzmB and NIRF nanosensors for fluorogenic cleavage assay as described above.

[0158] Detection of urinary reporters: Either one day before the first dose or 24 hours after the third dose of ^PD1 / ^CTLA4 treatment, FAM-labeled nanosensors were intratumorally administered. Urine was collected for three hours. FAM reporters were isolated from urine samples by immunoprecipitation using Dynabeads (ThermoFisher) decorated with anti- fluorescein isothiocyanate antibody (GeneTex) with elution in acetic acid (5% v / v) and neutralization in Tris buffer (2^M, pH 12). Sample fluorescence was measured using a Cytation5 plate reader (Biotek), and reporter concentrations were determined by using a known FAM ladder.

[0159] Toxicology: AND-gated nanosensors were intravenously injected twice, one week apart. Body temperature was measured by rectal probe. One week after the second injection, whole blood was collected in serum separator tube, incubated at room temperature for 30 minutes to allow for clotting, and centrifuged at 2500xg for 10 minutes at 4C, after which serum was isolated. Serum chemistry analysis was performed by Antech Diagnostics, and serum cytokines were analyzed using LEGENDplex assay (Biolegend).

[0160] Software and statistical analysis: Graphs were plotted and appropriate statistical analyses were conducted using GraphPad Prism (*P^<^0.05, **P^<^0.01, ***P^<^0.001, central values depict the means and error bars depict s.e.m.). At least 3 replicates were used for all statistical analyses. Measurements were taken from independent samples, using biological replicates when possible. NIRF images were analyzed using Image Studio (LI-COR). Flow cytometry data were analyzed using FlowJo X (FlowJo). Power analyses were performed using G*Power 3.1 (HHUD). Results

[0161] Asymmetric bi-labile cyclic peptide nanosensors implement AND-gate logic: It was postulated that a bi-labile cyclic peptide design could implement AND-gate logic if the release of the reporter occurs if and only if both substrates are cleaved (FIG. 1A). To test this design concept, the study synthesized a fluorogenic linear peptide (IEFDSG (SEQ ID NO: 10)) selective for granzyme B (GzmB) and its symmetric bi-labile cyclic peptide counterpart that contained two copies of the substrate. Both constructs made use of a fluorophore and quencher ^Attorney Docket No.10034-321WO1 FRET pair (5(6)-carboxyfluorescein and Tide Quencher 2, respectively) to allow monitoring of proteolysis by fluorimetry (FIGS. 2A-2B). Incubation of the fluorogenic linear substrate with recombinant GzmB resulted in a rapid increase in sample fluorescence (FIG.3A), which was confirmed to result from substrate cleavage by the appearance of two distinct mass fragments (1128.2 and 1072.2 Da fragments containing fluorophore and quencher, respectively) by mass spectrometry (FIG.3B). By contrast, whereas GzmB digestion of the bi- labile cyclic peptide likewise resulted in a rapid increase in sample fluorescence, the mass spectrum contained two fully cleaved fragments (1656.8 and 1729.0 Da), as well as a third species corresponding to a linearized form of the cyclic peptide with a mass shift of +18 Da (3367.8 Da compared to 3349.8 Da for the fully intact peptide; FIGS. 3C-3D), which was attributed to the cleavage of a single arm of the cyclic peptide. To further test that dual substrate cleavage is required for reporter release, the study replaced either substrate of the cyclic peptide with that of a substrate synthesized from d-amino acids to prevent proteolysis and found a significant reduction in sample fluorescence compared to the fully labile l-amino acid version of the cyclic peptide (FIGS. 3E-3F). These studies confirmed that reporter release from the cyclic peptide design requires cleavage of both substrates.

[0162] To confirm the ability of asymmetric cyclic peptides to perform AND logic, the study synthesized a bi-labile cyclic peptide containing the substrate sequence IEFDSG (SEQ ID NO: 10) and the substrate sequence APAALRAA (SEQ ID NO: 11) for which the study characterized had broad activity for matrix metalloproteinases (MMPs) and negligible activity for off-target proteases including GzmB, cathepsins, and caspases (FIGS. 1B-1C, FIGS. 4A- 4B). This FRET-labeled cyclic peptide was then incubated with either GzmB, MMP8, or both proteases, and it was found that both proteases were required for production of a reporter fragment detectable by mass spectrometry or fluorescence (FIG. 1D, FIGS. 5A-5B), confirming that cyclic peptides actuate AND-gate logic. Given the short circulation half-lives of free peptides in vivo, the study formulated AND-gated nanosensors by conjugating cyclic peptides to iron oxide nanoparticles (IONPs) using PEGylated crosslinkers. Nanocarriers have been shown to extend circulation time of free peptides and reduce renal clearance rates, and PEGylation limits uptake by the reticuloendothelial system. The study validated that peptide conjugation onto the surface of IONPs resulted in an increase in particle size and the appearance of an absorbance peak at ~500 nm due to the FRET pair (FIGS. 6A-6C). Using this design, the study confirmed that the AND-gated nanosensors recapitulated the biological truth table by producing the highest reporter signal only in the condition when GzmB activity was concomitant with the activity of MMPs such as MMP-1, -2, -8, -9, or -13 (FIGS.1E-1F). ^Attorney Docket No.10034-321WO1 The study further evaluated the modularity of the design by substituting the MMP substrate for fPRSG (SEQ ID NO: 15), a thrombin-responsive sequence (FIGS. 7A-7C), or replacing the GzmB substrate for AAN (SEQ ID NO: 12) or ASGPAGPA (SEQ ID NO: 13), which are selective for legumain and fibroblast activation protein (FAP), respectively (FIGS. 8A-8C). For all substrate combinations tested, the study confirmed the ability of the nanosensors to implement AND-gate logic.

[0163] Multivalent presentation of AND-gated peptides on a nanoparticle improves the catalytic efficiency of proteolysis: Whereas restricting the conformation of peptides such as via cyclization typically reduces their susceptibility to protease cleavage, multivalent substrate presentation on the surface of a nanoparticle produces a higher local substrate concentration that can enable an enzyme bound to one peptide to “hop” to other substrates on the same nanoparticle and increase cleavage rates. The study therefore sought to quantify potential differences in the catalytic efficiencies (i.e., kcat / Km) of proteolysis between the symmetrical bi-labile cyclic peptide of GzmB (IEFDSG (SEQ ID NO: 10)) and its linear form. The study determined catalytic efficiency based on release of the fluorescent reporter by using equimolar concentrations of the linear and cyclic peptides, such that the cyclic peptide required cleavage of twice as many substrate copies as the linear peptide for equivalent activation kinetics. Under the condition where either peptide was mixed in solution with GzmB, it was found that the catalytic efficiency of proteolysis for the cyclic peptide was ~85.2% less than that of the linear substrate (6.2 versus 41.9 M-1s-1, respectively). However, when either peptide was conjugated to the surface of IONPs to create a multivalent construct with ~40 peptides per IONP, the catalytic efficiency increased by ~21.7-fold for the cyclic peptide nanosensor compared to ~6.6-fold for the linear peptide nanosensor (FIGS. 9A-9B). This substantially reduced the difference in catalytic efficiency between the cyclic and linear peptides to ~51% (134.5 versus 276.2 M-1s-1, respectively), indicating that multivalent presentation of cyclic peptides can result in a larger increase in proteolytic efficacy compared to linear peptides.

[0164] To identify an optimal surface valency, cyclic peptides were conjugated to IONPs at increasing stoichiometric ratios to form nanosensors with valencies ranging from 2 to 197 peptides on average per IONP (Pearson’s r = 0.993 for 2 to 88 peptides per IONP; FIGS.10A- 10B). It was observed that initial cleavage velocities increased with valency to an apparent maximum value at ~44 peptides copies per IONP after which further increases in peptide valency decreased initial cleavage velocities, likely due to steric effects from overcrowding (FIG.9C, FIGS.10C-10D). Based on this data, the study selected a peptide:IONP ratio of ~44 for all future studies to maximize catalytic efficiency. To quantify the limit of detection of ^Attorney Docket No.10034-321WO1 protease sensing, the study exposed sensors to decreasing concentrations of GzmB and found that both linear and cyclic peptide sensors exhibited dose-dependent responses with the lowest detectable signal at 1.6 nM GzmB (FIGS. 9D-9E). Finally, the study tested the limit of detection of the GzmB and MMP AND-gated nanosensor and found that reporter fluorescence was dependent on the dose of both input proteases, with detectable signal at as low as 3.5 nM GzmB and 10 nM MMP9 (FIGS. 9F-9G). Taken together, the data showed that AND-gated nanosensors retain fast activation rates and high analytical sensitivity in response to dual protease inputs.

[0165] AND-gated nanosensors implement logic to detect T cell killing of tumor cells: The study next sought to evaluate the ability of the AND-gated nanosensors to differentiate physiologically unique conditions using cell-secreted proteases as inputs. CD8+ T cells express T cell receptors (TCRs) that recognize processed peptide antigens presented on class I major histocompatibility complexes (pMHCI). Upon TCR recognition of a cognate pMHCI, T cells degranulate to release effector molecules including GzmB that enter the target cell and initiate a cascade of events leading to cell death. Given the ubiquity of MMP secretion by cancer cells to promote angiogenesis, tumorigenesis, and metastasis, it was hypothesized that an AND- gated nanosensor that requires MMP and GzmB activity as inputs would respond only to the physiological condition where T cells are killing cancer cells and not others (e.g., conditions where tumor cells or T cells are in isolation).

[0166] To test this, the study collected media conditioned by either primary murine CD8+ T cells activated by ^CD3 and ^CD28 antibodies or MC38 colorectal cancer cells (FIG.11A), confirmed the presence of secreted GzmB or MMP9 by ELISA (FIGS. 11B-11C), and validated orthogonal activation of the GzmB and MMP linear substrates (FIGS.11D-11E). To test the AND-gated peptide against the biological truth table for GzmB and MMP9, the study used a combined sample containing conditioned media collected from both T cells and cancer cells and found that it led to ~3-fold higher activation of AND-gated peptides than incubation with medium collected from only T cells or only cancer cells (FIG. 11F). Based on these results, the study next assessed whether AND-gated nanosensors can selectively detect antitumor T cell killing apart from other co-culture conditions. The study mixed TCR- transgenic OT-1 CD8+ T cells with MC38 colorectal cancer cells that were either pulsed with the OT-1 cognate peptide antigen SIINFEKL (SEQ ID NO: 16) (OVA257-264) or AVYNFATC (SEQ ID NO: 17) (LCMV gp34-41) as a negative control peptide and verified antigen-specific cytotoxicity based on the release of lactate dehydrogenase (FIGS. 12A-12B). The study then collected media conditioned under four biological conditions: T cells only, tumor cells only, T ^Attorney Docket No.10034-321WO1 cells with tumor cells, and T cells with cognate antigen-pulsed tumor cells. Antigen-pulsed tumor cells were required for GzmB secretion by T cells, and all three conditions with tumor cells resulted in elevated MMP9 secretion (FIGS. 12C-12D). Incubation of AND-gated sensors led to significant activation from media conditioned by T cells with antigen-pulsed tumor cells within 2.5 minutes (P < 0.0001), with no significant activation from all other conditions after 30 minutes (P > 0.05; FIG.12E). Thus, the AND-gated sensors can implement logic using protease activity as inputs to discriminate T cell killing of antigen-expressing cancer cells.

[0167] AND-gated nanosensors detect responses to checkpoint blockade immunotherapy: Immune checkpoint blockade (ICB) therapy has transformed the treatment of cancer for patients across a broad range of malignancies, yet objective response rates remain below 40% for most cancer indications even with growing numbers of combination ICB trials. GzmB activity is a key driver of antitumor T cell responses, and several activity-based probes have been developed to detect GzmB activity as an early on-treatment biomarker of ICB responses. The study therefore tested whether AND-gated sensors that require activation by GzmB and MMPs can discriminate antitumor responses in a preclinical model of ICB.

[0168] C57BL / 6J mice bearing syngeneic MC38 flank tumors were treated with a triple drug combination of anti-programmed cell death protein 1 antibody (^PD1), anti-cytotoxic T lymphocyte-associated protein 4 antibody (^CTLA4), and interleukin 2 (IL-2). This therapy induced significant tumor regression (P < 0.0001) after three doses compared to mice treated with isotype control antibodies, with significant elevations in the frequency and number of GzmB+ CD8+ tumor-infiltrating lymphocytes (P = 0.0025 and 0.010, respectively; FIGS. 13A-13D, FIGS. 14A-14C, FIG. 15). To compare sensor activation in the tumor and non- tumor organs during ICB responses, the study designed AND-gated nanosensors labeled with a near-infrared fluorescence (NIRF) FRET pair (sulfo-Cyanine7 fluorophore, Tide Quencher 7WS quencher) such that the quencher is released upon dual cleavage, leading to NIRF at the site of activated NPs (FIG.13E). It was confirmed that NIRF AND-gated nanosensors produce elevated fluorescence only in the presence of both GzmB and MMPs and are activated by media conditioned during T cell killing of MC38 cancer cells (FIGS. 16A-16B). NIRF AND-gated nanosensors were then administered intratumorally in mice bearing MC38 tumors, one day after the third dose of ICB therapy, and the biodistribution of activated sensors was quantified three hours post-injection. In mice treated with control antibodies, the tumor fluorescence was ~4.9% of the total quantified fluorescent signal across the major organs, whereas in ICB-treated mice, the relative fluorescence was ~7-fold higher in tumor tissue (34.3%, P < 0.001; FIGS. ^Attorney Docket No.10034-321WO1 13F-13G, and FIGS.17A-17B). No significant changes in NIRF signal were observed in major organs (brain, heart, kidneys, liver, lungs, tumor-draining lymph nodes, spleen; FIGS. 13F- 13G). Tumor fluorescence produced by AND-gated nanosensors also differentiated ICB responses from resistant B2m– / –tumors, which escape recognition by CD8+ T cells due to lack of antigen presentation resulting in reduced GzmB activity (FIGS. 18A-18B). Therefore, AND-gated sensors detect protease activity in ICB-responding tumors.

[0169] While activatable probes that emit fluorescence to highlight residual disease during surgical resection are under clinical evaluation, an in vivo assay based on a fluorescent readout is not routinely used in the clinic. Therefore, the study sought to determine whether an approach based on the intratumoral injection of AND-gated nanosensors followed by distal quantification of the released reporter in the urine could be used to assess responses to ICB therapy. It has been previously demonstrated that protease-cleaved peptide fragments that are below the renal filtration size limit are rapidly secreted and concentrated into the urine. Therefore, the study designed AND-gated nanosensors to release a fluorophore-labeled fragment into urine that can then be purified by immunoprecipitation for ex vivo quantification by fluorimetry. To evaluate this approach, the AND-gated sensors were intratumorally administered in mice one day after the third dose of ICB therapy and quantified reporters in urine three hours post-injection (FIG.13H). Urinary reporter levels were significantly elevated in ICB-treated mice compared to both mice treated with isotype antibodies and ICB-treated mice bearing resistant B2m– / –tumors (FIG.13I). In addition, urinary reporters indicated tumor responses with high diagnostic accuracy (AUROC = 0.85 relative to isotype treatment, 1.00 relative to B2m– / –tumors; FIG. 13J). These results demonstrate that the local activation of AND-gated nanosensors can be quantified distally from urine samples to indicate ICB- mediated antitumor T cell responses.

[0170] AND-gate logic requires co-localized proteases to increase activation specificity: The activity of GzmB by itself is not a specific biomarker of antitumor responses as T cells secrete GzmB during other pathologies like viral infection, autoimmunity, and transplant rejection. However, it was postulated that AND-gated sensing will increase detection specificity by minimizing signal production at tissue sites that may have increased T cell activity but not concomitant upregulation of MMPs. To test this, the study made artificial tissue mixtures by spiking recombinant GzmB into MC38 tumor homogenates with high MMP levels or healthy tissue homogenates with low MMP levels and observed that AND-gated nanosensor activation was on average at least 1.45-fold higher in the tumor samples than all other non- tumor organ samples (FIGS. 19A-19B). The study further tested specificity in vivo using a ^Attorney Docket No.10034-321WO1 mouse model of acute viral infection by the PR8 influenza A virus. The study confirmed acute infection by a significant decrease in the body weight of infected mice (FIG. 20A, FIG. 21) and a significant increase in the number of GzmB-expressing CD8+ T cells in the bronchoalveolar lavage fluid and lung tissue 8 days after infection compared to naïve mice (FIGS. 20B-20C). The study then intravenously administered linear GzmB nanosensors and AND-gated nanosensors to separate cohorts of mice and quantified NIRF across the major organs. In animals that received the linear GzmB sensor, 3.0-fold higher fluorescence was observed in the excised lungs of infected mice compared to naïve mice (P < 0.0001), but in mice that received the AND-gated nanosensor, the NIRF signals were statistically identical across all tissues (P > 0.05; FIGS.20E-20F, and FIGS.22A-22B). These results indicate that AND-gated sensors have improved local tissue specificity for T cell activity.

[0171] Next, the study sought to determine whether AND-gated nanosensor activation requires two proteases to be co-localized, as opposed to a mechanism whereby partial activation of the nanosensor by a single protease at one tissue site is followed by trafficking of the nanosensor to a distal site before a second protease completes activation. The study assessed this by inoculating mice with bilateral MC38 flank tumors using a combination of wild-type (w.t.) tumors that respond to ICB therapy (i.e., MMP high and GzmB high) or treatment- resistant B2m– / –tumors (i.e., MMP high and GzmB low) (FIG. 20G). It was reasoned that if high NIRF signals were observed in both the local w.t. tumor and in distal B2m– / –tumors after intratumoral delivery of AND-gated nanosensors only to the local tumor, this condition would indicate a “false positive” signal resulting from GzmB cleavage in the local w.t. tumor followed by nanosensor trafficking to the distal B2m– / –tumor for MMP activation. By contrast, if a high NIRF signal in local w.t. tumors was observed with a low NIRF signal in distal B2m– / –tumors, this outcome would provide support for a co-localized protease activation mechanism. Experimentally, it was observed that the intratumoral injection of AND-gated nanosensors into local w.t. tumors resulted in significantly lower NIRF signals in distal B2m– / –tumors compared to distal w.t. tumors (P < 0.01), supporting the latter hypothesis. The study also tested the reverse condition using local B2m– / –tumors and observed increased NIRF signals in distal w.t. tumors (FIGS.20H-20I), which provided additional support.

[0172] Intravenously delivered AND-gated nanosensors detect antitumor immunity with increased specificity: The study evaluated whether AND-gated nanosensors result in improvements to on-tumor specificity compared to linear GzmB nanosensors upon intravenous administration. The study first assessed toxicity of AND-gated nanosensors after systemic delivery. The study performed two intravenous injections of the nanosensors in healthy mice a ^Attorney Docket No.10034-321WO1 week apart to enable assessment of adaptive immune responses caused by repeated dosing. No significant changes in body weight, temperature, serum metabolites, or inflammatory cytokines were observed, indicating that the nanosensors were well-tolerated under the conditions tested (FIGS.23A-23F). Next, the study systemically administered either linear GzmB nanosensors or AND-gated nanosensors in mice receiving ICB therapy or isotype antibodies. ICB resulted in a ~1.25-fold increase in tumor fluorescence relative to isotype treatment in mice that received the linear GzmB nanosensor (FIGS. 24A-24B), compared to a significant ~2.1-fold increase in relative tumor fluorescence for ICB-treated mice produced by the AND-gated nanosensor (P < 0.001; FIGS.25A-25B, and FIGS.26A-26C). The increased activation of the AND-gated sensor may be in part due to differences in stability as cyclization can reduce degradation of peptides by serum proteases. A significant increase in activation of AND-gated sensors was also observed in the tumor-draining lymph nodes (P < 0.05), with no significant changes in other major organs. Given that AND-gate logic may increase specificity by requiring tumor-associated MMP activity, the study compared the tumor selectivity of each sensor. AND-gated nanosensors had significantly higher activation in ICB-responding tumors than 87.5% of tested non-tumor organs, compared to 37.5% for linear GzmB nanosensors (TABLE 1). Consequently, AND-gated nanosensors had higher selectivity for on-tumor detection than linear GzmB nanosensors (FIG.25C, FIG.27). TABLE 1. Statistical analysis of tumor-to-organ selectivity for intravenously administered nanosensors (n = 6-7, one-way ANOVA with Dunnett’s post-test and correction for multiple comparisons).^Attorney Docket No.10034-321WO1

[0173] While ICB is an FDA-approved standalone first-line therapy for cancers like melanoma, lung, head and neck, colorectal, renal, and esophageal cancers, many approved indications for ICB are either in combination with chemotherapy or as a second-line treatment after chemotherapy. Chemotherapeutics can induce on- and off-tumor inflammation and remodeling, processes that are mediated in part by MMP activity. The study therefore evaluated whether AND-gated nanosensors maintain on-tumor specificity when applied to detect ICB responses after preconditioning with one dose of oxaliplatin, a third-generation platinum compound used in standard-of-care treatments for advanced colorectal cancer. While oxaliplatin followed by ICB therapy resulted in a significant ~2.7-fold increase in tumor fluorescence relative to treatment with oxaliplatin and isotype antibodies (P < 0.0001), activation in off-tumor organs was not observed for either of the cohorts treated with oxaliplatin when compared with mice receiving only isotype antibodies (FIG.25D, and FIGS.28A-28C), indicating that AND-gated nanosensors maintain on-tumor specificity after chemotherapy.

[0174] Finally, the study evaluated whether AND-gated nanosensors are selectively activated at sites where protease pairs are co-localized using a model of ICB therapy in tumor- bearing mice infected with PR8 influenza A. Infected mice with tumors that are not responsive to ICB (either via isotype treatment or B2m– / –resistance) present a condition where GzmB and MMP activities are both elevated but at different tissue sites (i.e., the lungs and tumor, respectively). Therefore, tumor responses to ICB represent the unique condition where both GzmB and MMP are active in the same tissue site. Upon intravenous administration of NIRF AND-gated nanosensors, sensor activation was not observed in the tumor, lungs, or other major organs (liver and spleen) of PR8-infected mice bearingtumors, or of infected mice bearing w.t. tumors and receiving isotype antibodies, when compared to uninfected, isotype- treated controls (FIGS.25E-25F, and FIGS.29A-29B). By contrast, ICB treatment of infected mice bearing w.t. tumors resulted in a significant increase in tumor fluorescence compared to all control groups, without statistical changes in the lungs, liver, or spleen. Collectively, this data supports that AND-gated nanosensors remain inactive when target proteases are expressed at different tissue sites and are selectively activated by co-localized proteases to discriminate anti-tumor T cell responses from off-tumor antiviral immunity. Discussion

[0175] Genetically engineered biocircuits have established the ability of synthetic logic to enable biologics and biosensors that compute and conditionally activate in tumors with high specificity. This work demonstrates the application of activity-based nanosensors that ^Attorney Docket No.10034-321WO1 implement AND-gate logic without genetic components to increase detection precision for assessment of antitumor immune responses. AND-gated nanosensors responsive to the combination of GzmB from cytotoxic T cells and MMPs from cancer cells can differentiate ICB-responsive tumors from resistant tumors by producing tissue-localized imaging reporters or distal urinary reporters, with reduced activation in tissues without co-localized protease expression such as in the lungs during acute influenza infection. While genetic circuits continue to drive major advances in cell therapies and diagnostics, clinical translation of cell-based systems for in vivo applications often involves the need to reduce the immunogenicity of foreign components, such as by humanizing synthetic receptors, and may have restricted accessibility for larger patient populations, driving the development of “off-the-shelf” therapies using allogeneic cells or in vivo manufacturing to bypass an ex vivo pipeline. As such, the use of cell- and gene-free nanomaterials to design biosensors with the capacity to carry out Boolean logic could represent a lower barrier for translation.

[0176] GzmB is an established hallmark of responses to ICB therapy but is also an effector of T cell responses during viral infection, autoimmunity, and transplant rejection. Cancer patients are more susceptible to severe comorbidities caused by infections like influenza and pneumonia while ICB therapies can reactivate antiviral T cells and are associated with a high incidence of immune-related adverse events (irAEs) caused by autoimmune activity, for which immunosuppressive treatments can increase susceptibility to opportunistic infections. This study demonstrates that AND-gate logic can improve the specificity of ICB response assessment by discriminating GzmB activity during antitumor responses from off-tumor immune responses in flu-infected lungs based on the co-localized activity of MMPs in tumors. In addition, while chemotherapy is commonly used in combination with or prior to ICB regimens and can induce MMP-mediated processes like wound repair and tissue remodeling, it was found that platinum chemotherapy using oxaliplatin did not induce off-tumor activation of AND-gated nanosensors, and oxaliplatin followed by ICB therapy resulted in lower endpoint tumor volumes and higher sensor signals in the tumor than with ICB alone. These results are consistent with evidence that immunogenic cell death caused by chemotherapy may sensitize tumors to ICB therapy and increase the antitumor immune response.

[0177] Approximately half of the more than 550 proteases encoded by the human genome are secreted and found in the extracellular space. By designing bi-labile cyclic peptides against proteases secreted by CD8 T cells and cancer cells, the study showed that AND-gated sensing can distinguish between distinct physiological states, identifying the unique condition of antigen-specific cytotoxic killing of tumor cells from among other conditions. Given this ^Attorney Docket No.10034-321WO1 demonstration of platform modularity using substrate substitutes for other tumor-relevant proteases such as legumain, thrombin, and FAP, it is envisioned that this approach could be extended to simultaneously monitor multiple cell types in the tumor microenvironment, particularly as clinical responses to immunotherapy depend not only on CD8 T cell responses but more broadly on the interplay between pro-inflammatory and immunosuppressive cell populations. For example, imaging probes have been described that bind to or are cleaved by proteases expressed by macrophages, neutrophils, and cancer-associated fibroblasts during cancer progression or antitumor responses. In light of the fact that the majority of proteases encoded by the human genome have endopeptidase activity, these results suggest that this design can be extended to sense proteases expressed by other cell types in addition to CD8 T cells and tumor cells.

[0178] Intratumoral assessment of drug response is emerging as a strategy to predict treatment outcomes and guide personalized therapy. For example, implanted microdevices have been designed to deliver microdoses of multiple drugs and sample tumor tissue such that, after device extraction by biopsy, drug response can be analyzed by molecular and histological profiling. Another approach leverages DNA-barcoded liposomes loaded with cancer drugs to identify effective anticancer agents by single-cell sequencing of biopsied tumor tissue. Device implantation and tissue retrieval can be aligned with existing clinical workflows and surgical timeframes to minimize invasive procedures. This study tested this strategy in a model by using local intratumoral delivery of AND-gated nanosensors to produce remote biomarkers in urine that are detectable by liquid biopsy without requiring extraction of tumor tissue. It was found that this approach allowed assessment of urinary signals within 3 hours of intratumoral injection, likely due to the improved peptide activation kinetics from multivalent presentation. ICB therapies have received approvals as a first-line treatment option for both superficial tumors (e.g., melanoma and head and neck cancer) and cancers originating from deeper tissues (e.g., non-small-cell lung cancer and renal cancer).

[0179] Overall, these results demonstrate AND-gated nanosensors implement synthetic logic without genetic circuitry to produce detection signals after activation by protease pairs. This study provides support that AND-gated nanosensors detect the spatial distribution of proteases, which may have implications for the localized detection of disease and drug responses at specific tissue sites. ^Attorney Docket No.10034-321WO1 Example 3: AND-gated protease-activated nanosensors for programmable detection of anti-tumor immunity

[0180] The forward design of biosensors that implement Boolean logic to improve detection precision primarily relies on programming genetic components to control transcriptional responses. However, cell- and gene-free nanomaterials programmed with logical functions may present lower barriers for clinical translation. A study reports the design of activity-based nanosensors that implement AND-gate logic without genetic parts via bi- labile cyclic peptides. These actuate by releasing a reporter if and only if cleaved by a specific pair of proteases. AND-gated nanosensors that detect the concomitant activity of the granzyme B protease secreted by CD8 T cells and matrix metalloproteinases overexpressed by cancer cells identify the unique condition of cytotoxic T cell killing of tumour cells. In mouse models, AND-gated nanosensors discriminate tumours that are responsive to immune checkpoint blockade therapy fromtumours that are resistant to it, minimize signals from tissues without co-localized protease expression including the lungs during acute influenza infection, and release a reporter locally in tissue or distally in the urine for facile detection.

[0181] Advances in synthetic biology have historically focused on the genetic circuit paradigm to assemble sense-and-respond biocircuits operating under transcriptional regulation. Sophisticated functions have been implemented in prokaryotic and eukaryotic cells with applications across cell therapies, drug delivery, molecular imaging and bio-sensors. For example, bacteria have been genetically engineered to detect clinically useful biomarkers or produce synthetic biomarkers in biofluids. In mammalian cells, tumour-antigen sensing circuits that use AND-, OR- or NOT-gate logic have enabled programmable control of T cell responses to reduce toxicity. While the gene circuit analogy remains at the forefront of development, cell- and gene-free biocircuits are gaining attention for their ability to lower barriers for clinical translation while retaining the ability to perform logical functions. For example, RNA-based switches that regulate protein translation in response to cell-type-specific biomarkers or small- molecule inducers could be delivered via lipid nanoparticles without genetic engineering. Similarly, protein circuits that do not primarily rely on transcriptional regulation have been engineered as logic gates, band-pass filters, and regulatory cascades for antigen sensing, protein secretion and activation of enzymes and transcription factors. Recent work also highlights the growing potential of synthetic nanomaterials that, despite being cell- and gene-free, can apply logic to increase detection precision, often using cell-secreted proteases. These include hairpin peptide prodrugs that digitize drug delivery based on protease activity, imaging probes such as ‘hub-and-spoke’ fluorogenic peptides that require proteolysis of multiple quenchers to ^Attorney Docket No.10034-321WO1 highlight tumour margins, and activity-based sensors that utilize protease promiscuity as an analogue framework to classify biological samples.

[0182] This study describes a cell- and gene-free design of AND-gated nanosensors that require a pair of proteases for activation, increasing specificity. AND-gated nanosensors are implemented using asymmetric bi-labile cyclic peptides that release reporters if and only if both flanking substrates are cleaved. The cyclic peptides are multivalently displayed on iron oxide nanoparticles (IONPs) to improve catalytic efficiency and tissue retention. In mouse models, it is demonstrated that AND-gated nanosensors activated by a combination of matrix metalloproteinases (MMPs) in tumours and granzyme B (GzmB) secreted by cytotoxic T cells can selectively detect anti-tumour responses during immune checkpoint blockade therapy (ICBT) while minimizing activation from off-tumour sites including the lungs during acute viral infection. Results

[0183] Cyclic peptide nanosensors implement AND-gate logic: It is postulated that a bi- labile cyclic peptide design could implement AND-gate logic by releasing a reporter only when both substrates are cleaved (FIG. 1A). To test this design, a GzmB-selective linear peptide (IEFDSG (SEQ ID NO: 10)) and a cyclic peptide containing two substrate copies were synthesized. Both constructs used a Förster resonance energy transfer (FRET) pair (fluorophore, 5(6)-FAM; quencher, TQ2) for monitoring proteolysis by fluorimetry (FIG.2A). Incubation with GzmB rapidly increased fluorescence of the linear substrate (FIG. 3A), resulting from cleavage based on the appearance of two mass fragments (FIG.3B). By contrast, whereas GzmB digestion of the cyclic peptide resulted in rapid fluorescence, mass analysis revealed two fully cleaved fragments and a third species corresponding to a linearized form of the cyclic peptide (shift of +18 Da; FIGS.3C-3D), which was attributed to cleavage of a single arm of the peptide. To further test that dual substrate cleavage is required for reporter release, either substrate was replaced with an isoform using d-amino acids to prevent proteolysis. This significantly reduced fluorescence compared with the fully labile l-amino acid cyclic peptide (P < 0.0001; FIGS.3E-3F), further confirming that reporter release requires cleavage of both substrates.

[0184] To confirm that asymmetric cyclic peptides perform AND-gate logic, this study synthesized a cyclic peptide containing the GzmB substrate and the substrate APAALRAA (SEQ ID NO: 11), whose linear peptide had broad activity for MMPs and negligible activity for GzmB and other off-target proteases (FIGS. 1B-1C and FIGS. 4A-4B). The study incubated this FRET-labelled cyclic peptide with GzmB, MMP8 or both proteases. Both ^Attorney Docket No.10034-321WO1 proteases were required to produce a reporter detectable by mass spectrometry or fluorescence (FIG. 1D and FIGS. 5A-5B), confirming AND-gate logic. Given the short circulation half- lives of free peptides, AND-gated nanosensors were formulated by conjugating cyclic peptides to IONPs—which can extend circulation and reduce renal clearance of peptides—using PEGylated crosslinkers to limit uptake by the reticuloendothelial system. Peptide conjugation to IONPs resulted in increased particle size and an absorbance peak at ~500 nm owing to the FRET pair (FIGS. 6A-6C). Using this design, the study confirmed that AND-gated nanosensors produced highest reporter signals when GzmB activity was concomitant with MMPs (FIGS.1E-1F). The study further evaluated the modularity of the design by substituting the MMP substrate with fPRSG (SEQ ID NO: 15), a thrombin-responsive sequence, or replacing the GzmB substrate with substrates cleavable by legumain (AAN (SEQ ID NO: 12)) or fibroblast activation protein (ASGPAGPA (SEQ ID NO: 13)). Nanosensors using all substrate combinations successfully implemented AND-gate logic (FIGS. 6A-6C and FIGS. 8A-8C).

[0185] Multivalent presentation improves efficiency of proteolysis: Whereas restricting the conformation of peptides via cyclization reduces susceptibility to proteolysis, multivalent presentation on a nanoparticle (NP) increases local substrate concentrations and can enable enzymes to ‘hop’ between substrates on an NP, increasing cleavage rates. This study therefore evaluated differences in the catalytic efficiencies (kcat / Km, where kcatis the catalytic rate constant and Kmis the Michaelis constant) of proteolysis between the symmetrical GzmB- cleavable cyclic peptide and its linear form. The catalytic efficiency was determined based on release of the fluorescent reporter (that is, the cyclic peptide requires cleavage of two substrate copies). It was found that the catalytic efficiency of GzmB for the cyclic peptide was ~85.2% less than the linear substrate (6.2 M−1s−1versus 41.9 M−1s−1). However, conjugating peptides to IONPs to create multivalent constructs with ~40 peptides per IONP increased the catalytic efficiency by ~21.7-fold for the cyclic peptide compared with ~6.6-fold for the linear peptide (FIGS. 9A-9B). This substantially reduced the difference in kinetics between the cyclic and linear peptides to ~51% (134.5 M−1s−1versus 276.2 M−1s−1), indicating that multivalent presentation of cyclic peptides can result in larger increases in proteolytic efficacy compared with linear peptides.

[0186] To identify an optimal surface valency, the study synthesized nanosensors with valencies ranging from 2 to 197 peptides per IONP (FIGS. 10A-10B). It was observed that initial cleavage velocities increased with valency to a maximum of ~44 peptides per IONP, after which further increases in valency decreased cleavage kinetics, likely from steric effects ^Attorney Docket No.10034-321WO1 of overcrowding (FIG. 9C and FIGS. 10C-10D). A valency of ~44 was selected for future studies. To quantify the limit of detection, sensors were exposed to decreasing concentrations of GzmB. Both linear and cyclic peptide nanosensors exhibited dose-dependent responses with lowest detectable signals at 1.6 nM GzmB (FIGS. 9D-9E2). Finally, the limit of detection of the GzmB / MMP AND-gated nanosensor was tested and it was found that reporter fluorescence was dependent on both proteases, with detectable signal at as low as 3.5 nM GzmB and 10 nM MMP9 (FIGS.9F-9G). This data indicates that AND-gated nanosensors retain fast activation rates and high analytical sensitivity to dual protease inputs.

[0187] AND-gated nanosensors detect T cell killing of tumour cells: It was next evaluated whether AND-gated nanosensors can differentiate physiologically unique conditions using cell-secreted proteases. Upon recognition of cells expressing a cognate antigen, CD8+T cells release effector molecules including GzmB that initiate a cascade of events, leading to target cell death. Given the ubiquity of MMP secretion by cancer cells to promote angiogenesis, tumorigenesis and metastasis, it was hypothesized that AND-gated nanosensors requiring both MMP and GzmB would respond only to the condition where T cells are killing cancer cells and not when only tumour cells or T cells are present. To test this, media conditioned by either activated mouse CD8+T cells or MC38 colorectal cancer cells was collected (FIG. 11A), the presence of secreted GzmB or MMP9 was confirmed by ELISA (FIGS. 11B-11C), and orthogonal activation of the GzmB and MMP linear substrates was validated (FIGS. 11D- 11E). The AND-gated peptide was tested against the biological truth table for GzmB and MMP9 and it was found that pooling media from T cells and cancer cells increased activation of AND-gated peptides ~3-fold compared with medium from T cells or cancer cells alone (FIG. 11F). Next, it was assessed whether AND-gated nanosensors differentiate anti-tumour T cell killing from other co-culture conditions. OT-1 CD8+T cells were mixed with MC38 cells pulsed with either the OT-1 cognate peptide antigen OVA257–264 or the non-cognate LCMV gp34–41 peptide and antigen-specific cytotoxicity was verified by release of lactate dehydrogenase (FIGS. 12A-12B). Media was then collected from T cells only, tumour cells only, T cells with tumour cells or T cells with cognate antigen-pulsed tumour cells. Antigen- pulsed tumour cells were required for GzmB secretion by T cells, and all conditions with tumour cells showed elevated MMP9 secretion (FIGS. 12C-12D). Incubation of AND-gated nanosensors led to significant activation from media conditioned by T cells with antigen-pulsed tumour cells within 2.5 min (P < 0.0001), with no significant activation from all other conditions after 30 min (P > 0.05; FIG. 12E). Thus, AND-gated nanosensors can process protease activity to discriminate T cell killing of cancer cells. ^Attorney Docket No.10034-321WO1

[0188] AND-gated nanosensors detect responses to ICBT: ICBT has transformed patient outcomes across many cancer types, yet objective response rates remain below 40% for most indications. GzmB is a key driver of anti-tumour responses, and several activity-based probes have been developed to assess ICBT responses using GzmB activity. Therefore it was tested whether AND-gated nanosensors discriminate anti-tumour responses in a preclinical model of ICBT.

[0189] Mice bearing syngeneic MC38 flank tumours were treated with a triple-drug combination of anti-PD-1 antibody, anti-CTLA43 antibody and IL-2. This therapy induced significant tumour regression after three doses compared with mice treated with isotype control antibodies (P < 0.0001), with significant elevations in the frequency (P = 0.0005) and number (P = 0.0349) of GzmB+CD8+tumour-infiltrating lymphocytes (FIGS. 30A-30D, FIGS. 14A- 14C, and FIG.15). To compare sensor activation in the tumour and non-tumour organs during ICBT responses, AND-gated nanosensors labelled with a near-infrared fluorescence (NIRF) FRET pair (sulfo-Cyanine7 and TQ7WS) were designed such that the quencher is released upon dual cleavage, leading to NIRF at the site of activated NPs (FIG. 30E). These NIRF AND-gated nanosensors produced elevated fluorescence only when both GzmB and MMPs were present and were activated by media conditioned during T cell killing of MC38 cancer cells (FIGS. 16A-16B). NIRF AND-gated nanosensors were administered intratumourally in mice bearing MC38 tumours, 1 day after the third ICBT dose, and the biodistribution of activated sensors was quantified 3 h post-injection. In mice treated with isotype antibodies, tumour fluorescence was ~4.9% of the total fluorescent signal across major organs, whereas in ICBT-treated mice, the relative fluorescence was ~7-fold higher in tumour tissue (34.3%, P < 0.001; FIGS.17A-17B). No significant changes in NIRF were observed in major organs (brain, heart, kidneys, liver, lungs, tumour-draining lymph nodes and spleen; P > 0.05; FIGS. 30F- 30G). Tumour fluorescence produced by AND-gated nanosensors also differentiated ICBT responses from resistant B2m– / –tumours, which lack antigen presentation and escape recognition by CD8+T cells, resulting in reduced GzmB activity (FIGS.33A-33B). Therefore, AND-gated nanosensors detect protease activity in ICBT-responding tumours.

[0190] While activatable fluorescent probes are under evaluation to highlight residual disease during surgical resection, fluorescence-based in vivo assays are not routinely used in the clinic. Therefore, it was evaluated whether intratumoural injection of AND-gated nanosensors followed by quantification of released reporters in urine could assess responses to ICBT, given that protease-cleaved peptide fragments are below the renal filtration size limit and rapidly concentrate in urine. AND-gated nanosensors were designed to release ^Attorney Docket No.10034-321WO1 fluorophore-labelled fragments into urine for purification by immunoprecipitation and quantification by fluorimetry. Three hours after intratumourally administering nanosensors, urinary reporters were significantly elevated in mice treated with ICBT compared with mice receiving isotype antibodies and ICBT-treated mice bearing B2m– / –tumours (P = 0.0220 and P = 0.0045; FIGS. 30H-30I). Urinary reporters indicated response with high diagnostic accuracy (AUROC = 0.85 versus isotype, 1.00 versusFIG. 30J). These results demonstrate that local activation of AND-gated nanosensors can be quantified distally in urine to indicate anti-tumour T cell responses.

[0191] AND logic increases specificity to co-localized proteases: T cells secrete GzmB not only during anti-tumour responses but also pathologies such as viral infection, autoimmunity and transplant rejection. However, it was postulated that AND-gated sensing will increase specificity by minimizing signal in tissues with T cell activity but without MMP upregulation. To test this, artificial tissue mixtures were made by spiking GzmB into MC38 tumour homogenates (MMP-high) or healthy tissue homogenates (MMP-low). Nanosensor activation was 1.45-fold higher in tumour samples than all other samples (FIGS. 19A-19B). Specificity in vivo was further tested using a mouse model of acute viral infection by PR8 influenza A. Infection was confirmed by decreased body weight (FIG. 31A and FIGS. 21A-21B) and increased GzmB+CD8+T cells in the bronchoalveolar lavage fluid (BALF) and lungs 8 days post-infection compared with naive mice (FIGS. 31B-31C). AND-gated or linear GzmB nanosensors were then intravenously administer to separate cohorts and NIRF was quantified in major organs. Linear GzmB nanosensors resulted in 3.0-fold higher fluorescence in the excised lungs of infected mice compared with naive mice (P < 0.0001), whereas NIRF signals were statistically identical across all tissues for mice receiving AND-gated nanosensors (P > 0.05; FIGS. 31E-31F and FIGS. 34A-34B). These results indicate that AND-gated nanosensors improve local tissue specificity for T cell activity.

[0192] Next, it was assessed whether AND-gated nanosensors require co-localized input proteases, as opposed to a mechanism whereby after partial activation by a protease at one tissue site, nanosensors traffic to a distal site where a second protease completes activation. This was assessed in mice bearing bilateral MC38 flank tumours using a combination of ICBT- responsive wild-type tumours (MMP-high / GzmB-high) or treatment-resistant B2m– / –tumours (MMP-high / GzmB-low) (FIG. 31G). It was reasoned that high NIRF signals in distal B2m– / –tumours after intratumoral delivery of nanosensors to local wild-type tumours would indicate ‘false positive’ signals, resulting from GzmB cleavage in wild-type tumours followed by MMP activation in distal B2m– / –tumours. By contrast, high NIRF signals in local wild-type tumours ^Attorney Docket No.10034-321WO1 and low NIRF signals in distal B2m– / –tumours would support a co-localized protease activation mechanism. Experimentally, significantly lower NIRF signals were observed in distaltumours compared with distal wild-type tumours (P < 0.01) after local injection in wild-type tumours, supporting the latter hypothesis. The reverse condition was also tested using local B2m– / –tumours and increased NIRF signals were observed in distal wild-type tumours (FIGS. 31H-31I), providing additional support.

[0193] Improved systemic specificity for anti-tumour immunity: It was evaluated whether AND-gated nanosensors improve on-tumour specificity compared with linear GzmB nanosensors upon intravenous administration. First toxicity of systemically delivered AND- gated nanosensors was assessed. Two intravenous injections were performed in healthy mice a week apart to assess inflammatory responses caused by repeated dosing. No significant changes in weight, temperature, serum metabolites or inflammatory cytokines were observed, indicating that the nanosensors were well tolerated under these conditions (P > 0.05; FIGS. 23A-23G). Next, linear GzmB nanosensors or AND-gated nanosensors were systematically administered in mice receiving ICBT or isotype antibodies. ICBT resulted in ~1.25-fold higher tumour fluorescence relative to isotype treatment using linear GzmB nanosensors (FIGS.35A- 35B), compared with a significant ~2.1-fold increase using AND-gated nanosensors (P < 0.001; Fig. 32A-32B and FIGS. 36A-36C). The increased activation with AND-gated nanosensors may be due to improved stability as cyclization can reduce peptide degradation by serum proteases. Activation of AND-gated nanosensors in the tumour-draining lymph nodes was also observed, with no significant changes in other organs (P > 0.05). The tumour selectivity of the sensors was further compared. AND-gated nanosensors had significantly higher activation (P < 0.05) in ICBT-responding tumours than 87.5% of tested non-tumour organs compared with 37.5% for linear GzmB nanosensors (Table 1), resulting in higher tumour selectivity for AND-gated nanosensors (FIG.32C and FIG.27).

[0194] While ICBT is approved by the US Food and Drug Administration (FDA) for first- line use in several cancer types, many approved indications for ICBT are either in combination with or following chemotherapy. Chemotherapeutics can induce off-tumour inflammation and remodeling, which are mediated in part by MMPs. Therefore it was evaluated whether AND- gated nanosensors maintain specificity for anti-tumour responses after preconditioning with one dose of oxaliplatin, a platinum compound used in standard-of-care treatments for colorectal cancer. While oxaliplatin followed by ICBT resulted in ~2.7-fold higher tumour fluorescence relative to treatment with oxaliplatin and isotype antibodies, no activation in off-tumour organs was observed for either cohort when compared with mice receiving only isotype antibodies ^Attorney Docket No.10034-321WO1 (FIG.32D and FIGS.37A-37C), indicating that AND-gated nanosensors maintain on-tumour specificity after chemotherapy.

[0195] Finally, it was evaluated whether AND-gated nanosensors selectively activate at sites where protease pairs are co-localized using a comorbidity model of ICBT response and PR8 influenza infection. Infected mice with tumours that are not responsive to ICBT (via isotype treatment or B2m– / –resistance) have elevated GzmB and MMP at different tissue sites (lungs and tumour, respectively). Therefore, ICBT-responsive tumours represent the unique tissue site where both proteases are co-localized. Upon intravenous administration, NIRF AND-gated nanosensors did not activate in the tumour, lungs, liver or spleen of PR8-infected mice bearing B2m– / –tumours, or of infected mice bearing wild-type tumours and receiving isotype antibodies, when compared with uninfected, isotype-treated controls (FIGS.32E-32F and FIGS. 38A-38B). By contrast, ICBT in infected mice bearing wild-type tumours resulted in significantly higher tumour fluorescence compared with all control groups (P < 0.05), without statistical changes in the lungs, liver or spleen. Collectively, the data support that AND- gated nanosensors remain inactive when target proteases are expressed in different tissues and selectively detect co-localized proteases to discriminate anti-tumour responses from off- tumour anti-viral immunity. Conclusion^

[0196] Genetically engineered biocircuits have enabled biosensors that conditionally activate in tumours with high specificity by implementing synthetic logic. The study demonstrates the application of activity-based nanosensors that implement AND-gate logic without genetic components to increase detection precision for anti-tumour immune responses. While ICBT can induce T cell immunity off-tumour via reactivation of anti-viral T cells, immune-related adverse events and opportunistic infections upon treatment of adverse events, the present study shows that AND-gate logic improves specificity to anti-tumour responses and minimizes detection of T cell-secreted GzmB without co-localized MMP activity during off- tumour anti-viral immunity. While genetic circuits continue to drive major advances in cell therapies and diagnostics, clinical translation of cell-based systems for in vivo applications often requires reducing immunogenicity of foreign components, such as by humanizing synthetic receptors, and may have restricted accessibility for larger patient populations, driving the development of ‘off-the-shelf’ allogeneic cell therapies and in vivo manufacturing to bypass ex vivo pipelines. As such, cell- and gene-free biosensors that implement logic present lower barriers for translation. ^Attorney Docket No.10034-321WO1

[0197] While the study focused on detection of T cell-mediated anti-tumour responses, multiple inflammatory and immunosuppressive cell types contribute to responses to immunotherapy. For example, imaging probes have been described that bind to or are cleaved by proteases expressed by macrophages, neutrophils and cancer-associated fibroblasts during cancer progression or anti-tumour responses.

[0198] In the present study, detection strategies were validated using both intra- tumoral and systemic delivery of AND-gated nanosensors. ICBT has received first-line approvals for both superficial tumours and cancers originating from deeper tissues. Looking forward, intratumoral assessment may be rapidly applied in an outpatient setting for superficial tumours, complementing imaging-based approaches such as computed tomography (CT) and positron emission tomography (PET). Systemic delivery may be applicable for cancers in deeper tissues, which could be assessed using orthotopic models. In addition, chemotherapy is commonly used in first-line combinations with or before second-line ICBT. While chemotherapy can induce MMP-mediated processes such as wound repair and tissue remodelling, AND-gated nanosensors maintained minimal off-tumour activation when platinum chemotherapy preceded ICBT while producing higher on-tumour signals, consistent with evidence that immunogenic cell death caused by chemotherapy may sensitize tumours to ICBT and increase anti-tumour responses.

[0199] Overall, the results demonstrate that AND-gated nanosensors implement synthetic logic without genetic circuitry to produce detection signals after activation by protease pairs, which has implications for localized detection of disease and drug responses at specific tissue sites. Methods^

[0200] Animals: Female mice (6–10 weeks old) were used at the outset of all experiments. OT-1 (C57BL / 6-Tg(TcraTcrb)1100Mjb / J) transgenic mice were bred in-house using breeding pairs purchased from The Jackson Laboratory ( Jax, 003831). C57BL / 6J (B6) mice were purchased from Jax (000664) and were used as recipients for all tumour models. A maximal tumour size of 1.5 cm in any dimension was used as an endpoint for all tumour models and not exceeded. All animal procedures in this study received ethical approval by the Georgia Tech Institutional Animal Care and Use Committee (protocol numbers KWONG-A100190, KWONG-A100191 and KWONG-A100193).

[0201] Peptide synthesis: Peptides were either synthesized in-house or ordered from a vendor (LifeTein, GenScript or CPC Scientific). In-house synthesis was performed by Fmoc solid-phase peptide synthesis on a Liberty Blue peptide synthesizer (CEM) using low-loading ^Attorney Docket No.10034-321WO1 rink amide resin (CEM). In brief, for each amino acid cycle, Fmoc deprotection was performed using 20% (v / v) 4-methylpiperidine in dimethylformamide, and amino acids (5 equivalents (eq.); Chem Impex) were coupled in the presence of 10 eq. diisopropylcarbodiimide (DIC) and 5 eq. Oxyma Pure. After synthesis, peptides were cleaved off resin using 92.5% trifluoroacetic acid, 2.5% water, 2.5% triisopropylsilane and 2.5% 3,6-dioxa-1,8-octane-dithiol for 30–45 min at 41 °C using a Razor (CEM). After cleavage, peptides were precipitated in ice-cold diethyl ether and vacuum dried overnight. Crude peptides were purified by reverse phase high- performance liquid chromatography (HPLC; 1260 Infinity II, Agilent) using a Zorbax C18 column (Agilent) using an elution gradient from 5% to 100% acetonitrile in 0.05% trifluoroacetic acid. Acetonitrile was removed from purified fractions by rotary evaporation, and samples were frozen overnight and lyophilized.

[0202] To make cyclic peptides, linear peptides were first synthesized with Lys(Alloc) near the C-terminus and Glu(OAllyl) at the N terminus. Fmoc-protected resin was then treated with phenylsilane and tetrakis(triphenylphosphine)palladium(0) in dichloromethane at 35 °C to restore Lys and Glu side chains, followed by cyclization using DIC and Oxyma.

[0203] For labelling with fluorophores and quenchers, resin-coupled peptides were N- terminally labelled with 5(6)-carboxyfluorescein (FAM) directly on Liberty Blue with DIC and Oxyma Pure after Fmoc deprotection. Alternatively, Fmoc deprotection was performed on Liberty Blue, and resin-coupled peptides were N-terminally labelled with Tide Quencher 7WS (TQ7) succinimidyl ester (AAT Bioquest) in dimethyl sulfoxide with 4–10 eq. triethylamine at 37 °C for 2 h. Peptides, which were synthesized with propargylglycine, were cleaved from resin and labelled with Tide Quencher 2 (TQ2) azide (FAM peptides; AAT Bioquest) or sulfo- Cyanine7 azide (Lumiprobe) by copper(I)-catalysed azide-alkyne cycloaddition (CuAAC). CuAAC reaction was performed using copper(II) sulfate, tris(3- hydroxypropyltriazolylmethyl)amine (THPTA), ascorbic acid and aminoguanidine in phosphate-buffered saline (PBS) at 37 °C overnight. Peptides were purified by HPLC after dye labelling.

[0204] Mass spectrometric analysis of synthetic peptides and cleavage products: Mass analysis of crude and purified peptides synthesized in-house was performed by liquid chromatography-mass spectrometry (LC / MS; 1260 Infinity II and InfinityLab LC / MSD, Agilent; OpenLab CDS ChemStation v2.7 software) on positive ion mode electrospray ionization (ESI / MS) using a Zorbax or Poroshell C18 column (Agilent) and an elution gradient from 5% to 100% acetonitrile in either 0.05% trifluoroacetic acid or 0.05% formic acid. Mass analysis of peptide cleavage products was performed by ESI / MS (Agilent) or by matrix- ^Attorney Docket No.10034-321WO1 assisted laser desorption / ionization mass spectrometry (MALDI-MS; Autoflex, Bruker) using an ^-cyano-4-hydroxycinnamic acid matrix. Before analysis, cleavage products were diluted to 75% acetonitrile and refrigerated for 30 min to precipitate proteins, followed by centrifugation at 4,000g for 5 min.

[0205] Nanosensor conjugation: Amine-functionalized IONPs were synthesized in-house as previously described and purified by fast-performance liquid chromatography (FPLC; AKTA Pure, Cytiva; Unicorn v7.11 software) using a Superdex 200 Increase 10-300 GL column, resulting in particles of ~20 nm average hydrodynamic diameter as determined by dynamic light scattering (Zetasizer, Malvern). For conjugation to peptides, NPs were labelled with SM(PEG)6 (ThermoFisher) for 2 h and then crosslinked to peptides containing free cysteines overnight. Reactions were performed in PBS (pH 7.2–7.4) at room temperature; 2 mM ethylenediaminetetraacetic acid was added to the peptide crosslinking reaction to reduce formation of disulfide bonds between peptides. Nanosensors were purified by FPLC. Nanosensor valency (that is, peptide-to-NP ratio) was quantified by ultraviolet–visible spectroscopy, using 400 nm for IONPs and 490 nm for FAM / TQ2-labelled peptides or 750 nm for Cy7 / TQ7-labelled peptides. All valency calculations were performed using optical density (OD) values below 2.0 to ensure linearity of the Beer–Lambert law. Samples were diluted in PBS to achieve OD <2.0 when necessary.

[0206] Cell culture: MC38 cells (gift from the National Cancer Institute and D. Vignali, University of Pittsburgh) and B2m− / −MC38 cells (developed in previous work) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PenStrep; ThermoFisher). Cells were grown to 70–90% confluence before being trypsinized and passaged with a split ratio of 20–40:1. For OT-1 T cells, OT-1 transgenic mice were killed, primary splenocytes were collected, and CD8 T cells were isolated (CD8a+T cell isolation kit, Miltenyi). T cells were activated for 48 h using anti-CD3e and anti-CD28 antibodies (BD Biosciences; clones 145-2C11 and 37.51, respectively) and cultured for up to 4 days at <3 million cells per ml, using T cell media (RPMI 1640 supplemented with 10% FBS, 1% PenStrep, 1% non-essential amino acids, 1% sodium pyruvate, 4 ppm (v / v) beta- mercaptoethanol and 30 units per ml IL-2 (Roche)).

[0207] Fluorogenic substrate assay: GzmB (Peprotech 140-03), thrombin (Thrb; Prolytix HCT-0020), fibroblast activation protein (FAP; R&D Systems 3715-SE), gamma- glutamyltransferase 1 (GGT1; R&D Systems 10977-GT), granzyme A (GZMA; Enzo Life Sciences ALX-201-118), ADAMTS1 (R&D Systems 2197-AD), tissue-type plasminogen activator (TPA; R&D Systems 7449-SE), urokinase-type plasminogen activator (UPA; R&D ^Attorney Docket No.10034-321WO1 Systems 1310-SE) and plasmin (PLG; Prolytix HCPM-0140) were diluted directly in PBS. MMPs (Enzo Life Sciences BML-AK013 and BML-AK014) were diluted in MMP activation buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.1 mM ZnCl2, 0.05% Brij-35, pH 7.4) and activated for 30 min at 37 °C before dilution in PBS. Legumain (LGN; R&D Systems 2058- CY) and cathepsins (CTSD, CTSE, CTSS; Enzo Life Sciences BML-SE199, R&D Systems 1294-AS, R&D Systems 1183-CY) were diluted in LGN activation buffer (100 mM NaOAc, 100 mM NaCl, pH 4.5; 20 mM DTT was added for CTSS) and activated at 37 °C before dilution in PBS to final pH ~5.5. Caspases (CASP1, CASP3, CASP8; Enzo Life Sciences BML-SE168, BML-SE169, BML-SE172) were diluted in caspase activation buffer (50 mM HEPES, 100 mM NaCl, 20 mM DTT, 2 mM EDTA, 0.1% Tween-20, 10% glycerol) and activated for 30 min at 37 °C before dilution in PBS. Conditioned media was isolated freshly from cultured cells by centrifugation at 1,000g for 5 min. Peptides or nanosensors were mixed with recombinant proteases or conditioned media and incubated at 37 °C. For peptides labelled with FAM and TQ2, sample fluorescence was measured by a Cytation 5 plate reader (Biotek; Gen5 software). For peptides labelled with Cy7 and TQ7, sample fluorescence was measured by Odyssey CLx Imager (LI-COR; ImageStudio v5.2 software). Peptide concentrations were 1– 10 ^M for free peptides in solution and 0.1–1 ^M for nanosensors. Protease concentrations were 50–250 nM unless otherwise specified.

[0208] T cell cytotoxicity assay: Five days after collecting OT-1 T cells, MC38 cells were trypsinized and incubated at 1 million cells per ml with 30 ^M antigen peptide (OVA or gp34) in OptiPro for 1 h at 37 °C. Antigen-pulsed MC38 cells were washed in T cell media, and 60,000 cells were seeded in a 96-well plate. After 4–6 h, OT-1 T cells were washed in T cell media, and 300,000 cells were added to the tumour cells (effector-to-target ratio = 5:1). After overnight incubation (12–16 h), conditioned media was isolated by centrifugation. Conditioned media was either used for fluorogenic substrate assay or analysed by lactate dehydrogenase assay (Abcam) or ELISA for GzmB (ThermoFisher) or MMP9 (Abcam).

[0209] Mouse models of cancer ICBT: B6 mice were subcutaneously inoculated into the left flank with 1 million MC38 cells orMC38 cells. Both left and right flanks were inoculated for the bilateral tumour model. Tumour burden (0.52 × length × width × depth) was monitored twice weekly, and treatment was initiated at 300–400 mm3. Anti-PD1 antibody (gift from G. Freeman, with the help of R. Ahmed of Emory University, clone 8H3; original source, Dana-Farber, not commercially available) and anti-CTLA4 antibody (BioXCell; clone 9H10) or matched IgG1 and IgG2b isotype controls (BioXCell; clones MOPC-21 and MPC-11, respectively) were intravenously administered by tail vein injection every 3 days for up to 4 ^Attorney Docket No.10034-321WO1 doses (0.2 mg of each antibody per dose). IL-2 (10 ^g) (PeproTech) was intraperitoneally administered to mice receiving anti-PD1 / anti-CTLA4 treatment on the same schedule. For chemotherapy, 0.1 mg of oxaliplatin (ThermoFisher J66586.MF) was intraperitoneally administered 3 days before initiation of ICBT. For tumour-bearing mice infected with PR8 influenza A, mice were infected as described below 1 day before initiation of ICBT. Flow cytometric analysis and intratumoral or intravenous injection of nanosensors for NIRF imaging or urinalysis were performed 1 day after the third treatment dose.

[0210] Mouse models of influenza A virus: B6 mice were intranasally inoculated with 30 plaque-forming units (p.f.u.) of purified H1N1 influenza A virus (PR8; strain A / PR / 8 / 34). Body weight was measured daily. Flow cytometric analysis and intravenous injection of nanosensors for NIRF imaging were performed 8 days post-infection.

[0211] Organ dissociation and flow cytometry analysis: For tumour-bearing mice, MC38 tumours were enzymatically and mechanically dissociated using a mouse tumour dissociation kit (Miltenyi) and a gentleMACS dissociator (Miltenyi), respectively. Tumour-infiltrating lymphocytes were isolated from the single-cell suspension using a density gradient with Percoll centrifugation media (GE Life Sciences) and RPMI 1640 at a 44:56 volume ratio, followed by red blood cell (RBC) lysis. For PR8-infected mice and naive controls, BALF was collected using 3 washes with 1 ml of PBS. Cells were collected from BALF by centrifugation, followed by RBC lysis. Lungs were enzymatically dissociated using collagenase type IV. Lymphocytes were isolated from the single-cell suspension using a 40:60 Percoll / RPMI density gradient, followed by RBC lysis.

[0212] For flow cytometry analysis, cells were first stained for surface markers in FACS buffer (1× DPBS, 2% FBS, 1 mM EDTA, 25 mM HEPES), followed by viability staining with LIVE / DEAD Fixable Near-IR dye (Invitrogen). Cells were fixed and permeabilized (eBioscience) before intracellular staining for GzmB in permeabilization buffer. Stained cells were analysed using an LSRFortessa (BD Biosciences; FACSDiva v8 software) or Aurora (Cytek Biosciences; SpectroFlo v3.2.1 software) flow cytometer. To determine absolute cell counts, CountBright Absolute Counting Beads (ThermoFisher) were added to samples immediately before analysis.

[0213] Antibody clones used were anti-CD45 (30-F11), anti-CD3 (145-2C11), anti-CD8 (53-6.7), anti-CD4 (RM4-5), anti-NK1.1 (PK136), anti-CD19 (6D5) and anti-GZMB (GB12). Anti-GZMB was purchased from ThermoFisher; all other antibodies were purchased from BioLegend. All antibodies were used for staining at 1:100 dilution from stock concentrations. ^Attorney Docket No.10034-321WO1

[0214] NIRF imaging of sensor activation in whole organs and homogenates: Three hours after injection of NIRF Cy7 / TQ7-labelled nanosensors, tumour-bearing or PR8-infected mice were euthanized, and tumour and organs (brain, lungs, heart, liver, kidneys, spleen, tumour- draining and non-tumour-draining lymph nodes) were isolated, rinsed in PBS and imaged by Odyssey CLx (LI-COR; ImageStudio v5.2 software). Alternatively, tumour and organs were isolated from mice that did not receive NIRF nanosensors and were dissociated using a FastPrep-24 homogenizer (MP Biomedicals) with beads (Lysing Matrix D, MP Biomedicals) in tissue protein extraction buffer (T-PER, ThermoFisher). Homogenate was isolated by centrifugation at 14,000g for 5 min and added to GzmB and NIRF nanosensors for fluorogenic cleavage assay as described above.

[0215] Detection of urinary reporters: Either 1 day before the first dose or 24 h after the third dose of anti-PD1 / anti-CTLA4 treatment, FAM-labelled nanosensors were intratumourally administered. Urine was collected for 3 h. FAM reporters were isolated from urine samples by immunoprecipitation using Dynabeads (ThermoFisher) decorated with anti- fluorescein isothiocyanate antibody (GeneTex GTX42751, clone 19F / 2A3, multiple lots used; 1 mg antibody per 25 mg Dynabeads) with elution in acetic acid (5% v / v) and neutralization in Tris buffer (2 M, pH 12). Sample fluorescence was measured using a Cytation 5 plate reader (Biotek), and reporter concentrations were determined by using a known FAM ladder.

[0216] Toxicology: AND-gated nanosensors were intravenously injected twice, 1 week apart. Body temperature was measured by rectal probe. One week after the second injection, whole blood was collected in a serum separator tube, incubated at room temperature for 30 min to allow for clotting and centrifuged at 2,500g for 10 min at 4 °C, after which serum was isolated. Serum chemistry analysis was performed by Antech Diagnostics, and serum cytokines were analysed using LEGENDplex assay (BioLegend).

[0217] Software and statistical analysis: Graphs were plotted and appropriate statistical analyses were conducted using GraphPad Prism v8 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; error bars depict mean ± s.e.m.). At least three replicates were used for all statistical analyses. Measurements were taken from independent samples, using biological replicates when possible. Data distribution was assumed to be normal, but this was not formally tested. Equal variances were confirmed using the F test to compare variances. NIRF images were analysed using ImageStudio v5.2 (LI-COR). Flow cytometry data were analysed using FlowJo X. No statistical methods were used to pre-determine sample sizes, but sample sizes are similar to those reported in previous publications. For all animal studies, animals were randomly assigned to various experimental groups, including random assignments for which ^Attorney Docket No.10034-321WO1 tumour type was inoculated (for example, wild-type or , which intervention was given (for example, ICBT or isotype), which nanosensor was administered (for example, AND-gate or linear GzmB), and whether mice were infected with PR8 or naive. For tumour models, a stratified block randomization strategy was used to ensure similar tumour burden across all cohorts. When feasible during in vivo studies (for example, tumour measurements, organ collection and urine collection), data collection was performed blinded by de-identifying mice with ear tag numbers that were matched to group allocation only after data collection and analysis. During other in vivo procedures and in vitro studies, blinding was not feasible as procedures were performed by individual investigators who required knowledge of the experimental groups to administer the allocated condition (for example, for in vivo studies, administration of allocated treatment or nanosensor; for in vitro studies, addition of allocated protease or cell population). After data collection, all data were analysed quantitatively without knowledge of group allocation, after which samples were matched to their group for data visualization and statistical analysis. No data were excluded from the analyses. EXAMPLE ASPECTS

[0218] Example 1: A protease activity sensor comprising the sequence: X3-X1-k-R1-kpP-X4-k-X5-R2-k-X2-K-X6(SEQ ID NO: 1), wherein: k is D-lysine; p is D-proline; X1and X2are amino acids crosslinked by their side chains; X3is a quencher or an acetyl group and X4is a reporter molecule, or X3is a reporter molecule and X4is null or a quencher; X5is null or cysteine-D-lysine (Ck); X6is glycinamide; and R1and R2are protease cleavage sites for the same or different proteases.

[0219] Example 2: The protease activity sensor of any examples herein, particularly Example 1, wherein the reporter molecule is a fluorophore, an isotopically encoded peptide, or an amino acid linked to a fluorophore or isotopically encoded peptide by its side chain.

[0220] Example 3: The protease activity sensor of any examples herein, particularly Examples 1-2, wherein i) X1is glutamate or aspartate and X2is lysine, or ii) X1is lysine and X2is glutamate or aspartate; and wherein X1and X2are crosslinked by their side chains to form an amide moiety.

[0221] Example 4: The protease activity sensor of any examples herein, particularly Example 3, wherein the protease activity sensor has a sequence selected from: X3-Ek-R1-kpP-X4-k-R2-kKK-X6(SEQ ID NO: 2), ^Attorney Docket No.10034-321WO1X3-Kk-R1-kpP-X4-kCk-R2-kEK-X6(SEQ ID NO: 5), wherein X3is a quencher or an acetyl group and X4is a reporter molecule, or X3is a reporter molecule and X4is a quencher.

[0222] Example 5: The protease activity sensor of any examples herein, particularly Example 3, wherein the protease activity sensor has a sequence selected from: X3-Ek-R1-kpPk-R2-kKK-X6(SEQ ID NO: 6), X3-Kk-R1-kpPk-R2-kEK-X6(SEQ ID NO: 7), X3-Ek-R1-kpPkCk-R2-kKK-X6(SEQ ID NO: 8), or X3-Kk-R1-kpPkCk-R2-kEK-X6(SEQ ID NO: 9), wherein X3is a reporter molecule.

[0223] Example 6: The protease activity sensor of any examples herein, particularly Examples 1-2, wherein X1and X2are both cysteine; and wherein X1and X2are crosslinked by their side chains to form a disulfide bridge.

[0224] Example 7: The protease activity sensor of any examples herein, particularly Examples 1-6, wherein R1and / or R2is cleavable by a protease that indicates presence of a disease or disorder.

[0225] Example 8: The protease activity sensor of any examples herein, particularly Example 7, wherein R1and / or R2is cleavable by a cancer-associated protease or tumor- associated protease.

[0226] Example 9: The protease activity sensor of any examples herein, particularly Example 8, wherein the cancer is breast cancer, and R1and / or R2comprises any one of SEQ ID NOS: 274-364; wherein the cancer is colon cancer, and R1and / or R2comprises any one of SEQ ID NOS: 365-387; wherein the cancer is small cell lung carcinoma, and R1and / or R2comprises any one of SEQ ID NOS: 388-407; wherein the cancer is ovarian cancer, and R1and / or R2comprises any one of SEQ ID NOS: 398-417; wherein the cancer is prostate cancer, and R1and / or R2comprises any one of SEQ ID NOS: 418-437; wherein the cancer is liver cancer, and R1and / or R2comprises SEQ ID NO: 73, SEQ ID NO: 109, SEQ ID NO: 123, SEQ ID NO: 133, SEQ ID NO: 156, SEQ ID NO: 171, SEQ ID NO: 216, SEQ ID NO: 254, SEQ ID NO: 266, or any one of SEQ ID NOS: 438-448; wherein the cancer is adenocarcinoma, and ^Attorney Docket No.10034-321WO1 R1and / or R2comprises SEQ ID NO: 166, SEQ ID NO: 265, or any one of SEQ ID NOS: 449- 466; or wherein the cancer is renal cancer, and R1and / or R2comprises any one of SEQ ID NOS: 467-486.

[0227] Example 10: The protease activity sensor of any examples herein, particularly Examples 1-9, wherein R1and / or R2is cleavable by a tissue-specific protease.

[0228] Example 11: The protease activity sensor of any examples herein, particularly Example 10, wherein the tissue is kidney tissue, and R1and / or R2comprises any one of SEQ ID NOS: 487-496; wherein the tissue is liver tissue, and R1and / or R2comprises any one of SEQ ID NOS: 497-506; wherein the tissue is lung tissue, and R1and / or R2comprises any one of SEQ ID NOS: 507-516; wherein the tissue is heart tissue, and R1and / or R2comprises any one of SEQ ID NOS: 517-526; wherein the tissue is blood, and R1and / or R2comprises any one of SEQ ID NOS: 527-536; wherein the tissue is spleen tissue, and R1and / or R2comprises any one of SEQ ID NOS: 537-546; wherein the tissue is lymph node tissue, and R1and / or R2comprises any one of SEQ ID NOS: 547-556; wherein the tissue is stomach tissue, and R1and / or R2comprises any one of SEQ ID NOS: 557-566; wherein the tissue is ovarian tissue, and R1and / or R2comprise any one of SEQ ID NOS: 567-576; wherein the tissue is uterine tissue, and R1and / or R2comprises any one of SEQ ID NOS: 577-586; wherein the tissue is mammary gland tissue, and R1and / or R2comprises any one of SEQ ID NOS: 587-596; wherein the tissue is prostate tissue, and R1and / or R2comprises any one of SEQ ID NOS: 597-606; wherein the tissue is testicular tissue, and the prostate-cleavable linker comprises any one of SEQ ID NOS: 607-616; wherein the tissue is intestinal tissue, and R1and / or R2comprises any one of SEQ ID NOS: 617-626; wherein the tissue is bladder tissue, and R1and / or R2comprises any one of SEQ ID NOS: 627-636; wherein the tissue is brain tissue, and R1and / or R2comprises any one of SEQ ID NOS: 637-646; or wherein the tissue is thymic tissue, and R1and / or R2comprises any one of SEQ ID NOS: 647-656.

[0229] Example 12: The protease activity sensor of any examples herein, particularly Examples 1-11, wherein R1and / or R2is cleavable by a protease expressed by an immune cell during an immune response to a disease or disorder.

[0230] Example 13: The protease activity sensor of any examples herein, particularly Example 12, wherein R1and / or R2is cleavable by a protease expressed by an immune cell during an anti-tumor response.

[0231] Example 14: The protease activity sensor of any examples herein, particularly Examples 1-13, wherein R1and / or R2is cleaved by CASP1 and comprises any one of SEQ ID NOS: 25-34; wherein R1and / or R2is cleaved by CASP3 and comprises any one of SEQ ID ^Attorney Docket No.10034-321WO1 NOS: 35-44; wherein R1and / or R2is cleaved by CASP8 and comprises any one of SEQ ID NOS: 45-54; wherein R1and / or R2is cleaved by CTSD and comprises any one of SEQ ID NOS: 55-64; wherein R1and / or R2is cleaved by CTSE and comprises any one of SEQ ID NOS: 65-74; wherein R1and / or R2is cleaved by CTSG and comprises any one of SEQ ID NOS: 75-83; wherein R1and / or R2is cleaved by CTSS and comprises any one of SEQ ID NOS: 84-93; wherein R1and / or R2is cleaved by thrombin and comprises any one of SEQ ID NOS: 94-103; wherein R1and / or R2is cleaved by FAP and comprises any one of SEQ ID NOS: 104- 113; wherein R1and / or R2is cleaved by FXIA and comprises any one of SEQ ID NOS: 114- 123; wherein R1and / or R2is cleaved by GGT1 and comprises any one of SEQ ID NOS: 124- 133; wherein R1and / or R2is cleaved by GZMA and comprises any one of SEQ ID NOS: 134- 143; wherein R1and / or R2is cleaved by GZMB and comprises any one of SEQ ID NOS: 144- 153; wherein R1and / or R2is cleaved by H20S and comprises any one of SEQ ID NOS: 154- 163; wherein R1and / or R2is cleaved by KLK2 and comprises any one of SEQ ID NOS: 164- 173; wherein R1and / or R2is cleaved by MMP1 and comprises any one of SEQ ID NOS: 174- 183; wherein R1and / or R2is cleaved by MMP10 and comprises any one of SEQ ID NOS: 184- 193; wherein R1and / or R2is cleaved by MMP12 and comprises any one of SEQ ID NOS: 194- 203; wherein R1and / or R2is cleaved by MMP13 and comprises any one of SEQ ID NOS: 204- 213; wherein R1and / or R2is cleaved by MMP2 and comprises any one of SEQ ID NOS: 214- 223; wherein R1and / or R2is cleaved by MMP3 and comprises any one of SEQ ID NOS: 224- 233; wherein R1and / or R2is cleaved by MMP8 and comprises any one of SEQ ID NOS: 234- 243; wherein R1and / or R2is cleaved by MMP9 and comprises any one of SEQ ID NOS: 244- 253; wherein R1and / or R2is cleaved by TPA and comprises any one of SEQ ID NOS: 254- 263; or wherein R1and / or R2is cleaved by UPA and comprises any one of SEQ ID NOS: 264- 273.

[0232] Example 15: The protease activity sensor of any examples herein, particularly Examples 1-14, wherein R1and / or R2are each independently selected from IEFDSG (SEQ ID NO: 10), APAALRAA (SEQ ID NO: 11), AAN (SEQ ID NO: 12), ASGPAGPA (SEQ ID NO: 13), PAALRA (SEQ ID NO: 14), fPRSG (SEQ ID NO: 15), LVXXXSG (SEQ ID NO: 19), LVSPRSG (SEQ ID NO: 20), LVSFPSG (SEQ ID NO: 21), LVQNLSG (SEQ ID NO: 22), LVPRGSG (SEQ ID NO: 23), and PLGLAG (SEQ ID NO: 24).

[0233] Example 16: The protease activity sensor of any examples herein, particularly Examples 1-15, wherein R1and R2have different sequences that are cleaved by the same protease. ^Attorney Docket No.10034-321WO1

[0234] Example 17: The protease activity sensor of any examples herein, particularly Examples 1-15, wherein R1and R2have the same sequence.

[0235] Example 18: A nanoparticle comprising a plurality of the protease activity sensors of any examples herein, particularly Examples 1-17 anchored to a surface of said nanoparticle.

[0236] Example 19: The nanoparticle of any examples herein, particularly Example 18, wherein the nanoparticle comprises a plurality of thiol-reactive moieties on its surface; wherein X5is Ck; and wherein the thiol-reactive moieties are crosslinked to the cysteine in X5, thereby anchoring the plurality of protease activity sensors to the surface of the nanoparticle.

[0237] Example 20: The nanoparticle of any examples herein, particularly Example 19, wherein the plurality of thiol-reactive moieties comprise iodoacetyl, maleimide, or any combination thereof.

[0238] Example 21: The nanoparticle of any examples herein, particularly Examples 18- 20, wherein the nanoparticle comprises from about 2 to about 200 protease activity sensors anchored on its surface.

[0239] Example 22: The nanoparticle of any examples herein, particularly Examples 18- 21, wherein, upon cleavage of both R1and R2, the reporter molecule remains anchored to the surface of the nanoparticle; and wherein X3is a quencher or an acetyl group and X4is the reporter molecule.

[0240] Example 23: The nanoparticle of any examples herein, particularly Examples 18- 21, wherein, upon cleavage of both R1and R2, the reporter molecule is released from the nanoparticle.

[0241] Example 24: The nanoparticle of any examples herein, particularly Example 23, wherein X3is the reporter molecule and X4is null or a quencher.

[0242] Example 25: A method of detecting presence of a protease, the method comprising: a) providing the protease activity sensor of any examples herein, particularly Examples 1-17 or the nanoparticle of any examples herein, particularly Examples 18-24 to a sample or a subject, wherein R1and R2are protease cleavage sites for the same protease; and b) detecting presence or absence of the reporter molecule in the sample, the subject, or a sample taken from the subject, thereby detecting presence or absence of the protease; wherein presence of the reporter molecule indicates presence of the protease; and wherein absence of the reporter molecule indicates absence of the protease.

[0243] Example 26: The method of any examples herein, particularly Example 25, wherein step a) further comprises: i) providing the protease activity sensor to the subject; and ii) collecting a biological fluid sample from the subject; and wherein step b) further comprises ^Attorney Docket No.10034-321WO1 detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the protease.

[0244] Example 27: The method of any examples herein, particularly Example 25, wherein step a) further comprises providing the nanoparticle to the subject, wherein upon cleavage of both R1and R2, the reporter molecule remains anchored to the surface of the nanoparticle; and wherein step b) further comprises imaging the subject to detect presence or absence of the reporter molecule, thereby locally detecting presence or absence of the protease.

[0245] Example 28: The method of any examples herein, particularly Example 25, wherein step a) further comprises: i) providing the nanoparticle to the subject, wherein, upon cleavage of both R1and R2, the reporter molecule is released from the nanoparticle; and ii) collecting a biological fluid sample from the subject; and wherein step b) further comprises detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the protease.

[0246] Example 29: The method of any examples herein, particularly Examples 25-28, wherein presence of protease indicates presence and / or severity of a disease or disorder; and wherein the method is used to determine if the subject has said disease or disorder and / or progression of said disease or disorder.

[0247] Example 30: The method of any examples herein, particularly Examples 25-29, wherein presence of the protease indicates an immune response to a disease or disorder; and wherein the method is used to determine responsiveness of said disease or disorder to a treatment.

[0248] Example 31: The method of any examples herein, particularly Example 30, wherein the treatment is an immunotherapy.

[0249] Example 32: The method of any examples herein, particularly Examples 30-31, wherein the disease or disorder is cancer.

[0250] Example 33: The method of any examples herein, particularly Example 32, wherein the subject has a tumor; and wherein step a) further comprises injecting the protease activity sensor or the nanoparticle into said tumor.

[0251] Example 34: A method of detecting presence of two proteases, the method comprising: a) providing the protease activity sensor of any examples herein, particularly Examples 1-17 or the nanoparticle of any examples herein, particularly Examples 18-24 to a sample or a subject, wherein R1and R2are protease cleavage sites for different proteases; and b) detecting presence or absence of the reporter molecule in the sample, the subject, or a sample taken from the subject, thereby detecting presence or absence of the two proteases; wherein ^Attorney Docket No.10034-321WO1 presence of the reporter molecule indicates presence of both of the two proteases; and wherein absence of the reporter molecule indicates absence of one or both of the two proteases.

[0252] Example 35: The method of any examples herein, particularly Example 34, wherein step a) further comprises: i) providing the protease activity sensor to the subject; and ii) collecting a biological fluid sample from the subject; and wherein step b) further comprises detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the two proteases.

[0253] Example 36: The method of any examples herein, particularly Example 34, wherein step a) further comprises providing the nanoparticle to the subject, wherein upon cleavage of both R1and R2, the reporter molecule remains anchored to the surface of the nanoparticle; and wherein step b) further comprises imaging the subject to detect presence or absence of the reporter molecule, thereby locally detecting presence or absence of the two proteases.

[0254] Example 37: The method of any examples herein, particularly Example 34, wherein step a) further comprises: i) providing the nanoparticle to the subject, wherein, upon cleavage of both R1and R2, the reporter molecule is released from the nanoparticle; and ii) collecting a biological fluid sample from the subject; and wherein step b) further comprises detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the two proteases.

[0255] Example 38: The method of any examples herein, particularly Examples 34-37, wherein presence of both of the two proteases indicates presence and / or severity of a disease or disorder; and wherein the method is used to determine if the subject has said disease or disorder and / or progression of said disease or disorder.

[0256] Example 39: The method of any examples herein, particularly Examples 34-38, wherein presence of both of the two protease indicates an immune response to a disease or disorder; and wherein the method is used to determine responsiveness of said disease or disorder to a treatment.

[0257] Example 40: The method of any examples herein, particularly Example 39, wherein the treatment is an immunotherapy.

[0258] Example 41: The method of any examples herein, particularly Examples 39-40, wherein the disease or disorder is cancer.

[0259] Example 42: The method of any examples herein, particularly Example 41, wherein the subject has a tumor; and wherein step a) further comprises injecting the protease activity sensor or the nanoparticle into said tumor. ^Attorney Docket No.10034-321WO1

[0260] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein. Reference List Aggarwal, S. et al. Fibroblast activation protein peptide substrates identified from human collagen I derived gelatin cleavage sites. Biochemistry 47, 1076–1086 (2008). Algar, W. R. et al. Proteolytic activity at quantum dot-conjugates: kinetic analysis reveals enhanced enzyme activity and localized interfacial “hopping”. Nano Lett. 12, 3793–3802 (2012). Amaria RN, et al. Neoadjuvant immune checkpoint blockade in high-risk resectable melanoma. Nat Med 2018, 24(11): 1649-1654. Badeau BA, et al. Engineered modular biomaterial logic gates for environmentally triggered therapeutic delivery. Nature Chemistry 2018, 10(3): 251-258. Binnewies, M. et al. Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat. Med.24, 541–550 (2018). Brophy JAN, Voigt CA. 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Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease. Nat. Biotechnol.31, 63–70 (2013). ^Attorney Docket No.10034-321WO1 Kwong, G. A. et al. Synthetic biomarkers: a twenty-first century path to early cancer detection. Nat. Rev. Cancer 21, 655–668 (2021). Kyi, C., et al. Opportunistic infections in patients treated with immunotherapy for cancer. J. Immunother. Cancer 2, 19 (2014). Lin KY, et al. Nanoparticles That Sense Thrombin Activity As Synthetic Urinary Biomarkers of Thrombosis. ACS Nano 2013, 7(10): 9001-9009. Liu Z, et al. Legumain protease-activated TAT-liposome cargo for targeting tumours and their microenvironment. Nature Communications 2014, 5(1): 4280. López-Otín C, Matrisian LM. Emerging roles of proteases in tumour suppression. Nature Reviews Cancer 2007, 7(10): 800-808. Lord, S. J., et al. Granzyme B: a natural born killer. Immunol. Rev.193, 31–38 (2003). Mac, Q. D. et al. 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T Cells Expressing CD19 / CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells. Cancer Immunology Research 2016, 4(6): 498. Zaorsky NG, et al. Causes of death among cancer patients. Annals of Oncology 2017, 28(2): 400-407. Zhang Y, et al. Activatable Polymeric Nanoprobe for Near-Infrared Fluorescence and Photoacoustic Imaging of T Lymphocytes. Angewandte Chemie International Edition 2021, 60(11): 5921-5927. ^Attorney Docket No.10034-321WO1 Zhang, X. et al. Hepatitis B virus reactivation in cancer patients with positive hepatitis B surface antigen undergoing PD-1 inhibition. J. Immunother. Cancer 7, 322 (2019). Zhao, N. et al. In vivo measurement of granzyme proteolysis from activated immune cells with PET. ACS Cent. Sci.7, 1638–1649 (2021). Zhu, I. et al. Modular design of synthetic receptors for programmed gene regulation in cell therapies. Cell 185, 1431–1443.e1416 (2022). Zhuang, Q., et al. Deconvolving multiplexed protease signatures with substrate reduction and activity clustering. PLoS Comput. Biol.15, e1006909 (2019). ^Attorney Docket No.10034-321WO1 SEQUENCES^Attorney Docket No.10034-321WO1 PROTEASE CLEAVABLE LINKERS^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^Attorney Docket No.10034-321WO1^

Claims

Attorney Docket No.10034-321WO1 CLAIMS 1. A protease activity sensor comprising the sequence: X3-X1-k-R1-kpP-X4-k-X5-R2-k-X2-K-X6(SEQ ID NO: 1), wherein: k is D-lysine; p is D-proline; X1and X2are amino acids crosslinked by their side chains; X3is a quencher or an acetyl group and X4is a reporter molecule, or X3is a reporter molecule and X4is null or a quencher; X5is null or cysteine-D-lysine (Ck); X6is glycinamide; and R1and R2are protease cleavage sites for the same or different proteases.

2. The protease activity sensor of claim 1, wherein the reporter molecule is a fluorophore, an isotopically encoded peptide, or an amino acid linked to a fluorophore or isotopically encoded peptide by its side chain.

3. The protease activity sensor of any one of claims 1-2, wherein i) X1is glutamate or aspartate and X2is lysine, or ii) X1is lysine and X2is glutamate or aspartate; and wherein X1and X2are crosslinked by their side chains to form an amide moiety.

4. The protease activity sensor of claim 3, wherein the protease activity sensor has a sequence selected from:wherein X3is a quencher or an acetyl group and X4is a reporter molecule, or X3is a reporter molecule and X4is a quencher. ^Attorney Docket No.10034-321WO1 5. The protease activity sensor of claim 3, wherein the protease activity sensor has a sequence selected from:wherein X3is a reporter molecule.

6. The protease activity sensor of any one of claims 1-2, wherein X1and X2are both cysteine; and wherein X1and X2are crosslinked by their side chains to form a disulfide bridge.

7. The protease activity sensor of any one of claims 1-6, wherein R1and / or R2is cleavable by a protease that indicates presence of a disease or disorder.

8. The protease activity sensor of claim 7, wherein R1and / or R2is cleavable by a cancer- associated protease or tumor-associated protease.

9. The protease activity sensor of claim 8, wherein the cancer is breast cancer, and R1and / or R2comprises any one of SEQ ID NOS: 274-364; wherein the cancer is colon cancer, and R1and / or R2comprises any one of SEQ ID NOS: 365-387; wherein the cancer is small cell lung carcinoma, and R1and / or R2comprises any one of SEQ ID NOS: 388-407; wherein the cancer is ovarian cancer, and R1and / or R2comprises any one of SEQ ID NOS: 398-417; wherein the cancer is prostate cancer, and R1and / or R2comprises any one of SEQ ID NOS: 418-437; wherein the cancer is liver cancer, and R1and / or R2comprises SEQ ID NO: 73, SEQ ID NO: 109, SEQ ID NO: 123, SEQ ID NO: 133, SEQ ID NO: 156, SEQ ID NO: 171, SEQ ID NO: 216, SEQ ID NO: 254, SEQ ID NO: 266, or any one of SEQ ID NOS: 438-448; ^Attorney Docket No.10034-321WO1 wherein the cancer is adenocarcinoma, and R1and / or R2comprises SEQ ID NO: 166, SEQ ID NO: 265, or any one of SEQ ID NOS: 449-466; or wherein the cancer is renal cancer, and R1and / or R2comprises any one of SEQ ID NOS: 467-486.

10. The protease activity sensor of any one of claims 1-9, wherein R1and / or R2is cleavable by a tissue-specific protease.

11. The protease activity sensor of claim 10, wherein the tissue is kidney tissue, and R1and / or R2comprises any one of SEQ ID NOS: 487-496; wherein the tissue is liver tissue, and R1and / or R2comprises any one of SEQ ID NOS: 497-506; wherein the tissue is lung tissue, and R1and / or R2comprises any one of SEQ ID NOS: 507-516; wherein the tissue is heart tissue, and R1and / or R2comprises any one of SEQ ID NOS: 517-526; wherein the tissue is blood, and R1and / or R2comprises any one of SEQ ID NOS: 527-536; wherein the tissue is spleen tissue, and R1and / or R2comprises any one of SEQ ID NOS: 537-546; wherein the tissue is lymph node tissue, and R1and / or R2comprises any one of SEQ ID NOS: 547-556; wherein the tissue is stomach tissue, and R1and / or R2comprises any one of SEQ ID NOS: 557-566; wherein the tissue is ovarian tissue, and R1and / or R2comprise any one of SEQ ID NOS: 567-576; wherein the tissue is uterine tissue, and R1and / or R2comprises any one of SEQ ID NOS: 577-586; wherein the tissue is mammary gland tissue, and R1and / or R2comprises any one of SEQ ID NOS: 587-596; wherein the tissue is prostate tissue, and R1and / or R2comprises any one of SEQ ID NOS: 597-606; wherein the tissue is testicular tissue, and the prostate-cleavable linker comprises any one of SEQ ID NOS: 607-616; ^Attorney Docket No.10034-321WO1 wherein the tissue is intestinal tissue, and R1and / or R2comprises any one of SEQ ID NOS: 617-626; wherein the tissue is bladder tissue, and R1and / or R2comprises any one of SEQ ID NOS: 627-636; wherein the tissue is brain tissue, and R1and / or R2comprises any one of SEQ ID NOS: 637-646; or wherein the tissue is thymic tissue, and R1and / or R2comprises any one of SEQ ID NOS: 647-656.

12. The protease activity sensor of any one of claims 1-11, wherein R1and / or R2is cleavable by a protease expressed by an immune cell during an immune response to a disease or disorder.

13. The protease activity sensor of claim 12, wherein R1and / or R2is cleavable by a protease expressed by an immune cell during an anti-tumor response.

14. The protease activity sensor of any one of claims 1-13, wherein R1and / or R2is cleaved by CASP1 and comprises any one of SEQ ID NOS: 25-34; wherein R1and / or R2is cleaved by CASP3 and comprises any one of SEQ ID NOS: 35-44; wherein R1and / or R2is cleaved by CASP8 and comprises any one of SEQ ID NOS: 45-54; wherein R1and / or R2is cleaved by CTSD and comprises any one of SEQ ID NOS: 55-64; wherein R1and / or R2is cleaved by CTSE and comprises any one of SEQ ID NOS: 65-74; wherein R1and / or R2is cleaved by CTSG and comprises any one of SEQ ID NOS: 75-83; wherein R1and / or R2is cleaved by CTSS and comprises any one of SEQ ID NOS: 84-93; wherein R1and / or R2is cleaved by thrombin and comprises any one of SEQ ID NOS: 94-103; wherein R1and / or R2is cleaved by FAP and comprises any one of SEQ ID NOS: 104-^Attorney Docket No.10034-321WO1 wherein R1and / or R2is cleaved by FXIA and comprises any one of SEQ ID NOS: 114-123; wherein R1and / or R2is cleaved by GGT1 and comprises any one of SEQ ID NOS: 124-133; wherein R1and / or R2is cleaved by GZMA and comprises any one of SEQ ID NOS: 134-143; wherein R1and / or R2is cleaved by GZMB and comprises any one of SEQ ID NOS: 144-153; wherein R1and / or R2is cleaved by H20S and comprises any one of SEQ ID NOS: 154-163; wherein R1and / or R2is cleaved by KLK2 and comprises any one of SEQ ID NOS: 164-173; wherein R1and / or R2is cleaved by MMP1 and comprises any one of SEQ ID NOS: 174-183; wherein R1and / or R2is cleaved by MMP10 and comprises any one of SEQ ID NOS: 184-193; wherein R1and / or R2is cleaved by MMP12 and comprises any one of SEQ ID NOS: 194-203; wherein R1and / or R2is cleaved by MMP13 and comprises any one of SEQ ID NOS: 204-213; wherein R1and / or R2is cleaved by MMP2 and comprises any one of SEQ ID NOS: 214-223; wherein R1and / or R2is cleaved by MMP3 and comprises any one of SEQ ID NOS: 224-233; wherein R1and / or R2is cleaved by MMP8 and comprises any one of SEQ ID NOS: 234-243; wherein R1and / or R2is cleaved by MMP9 and comprises any one of SEQ ID NOS: 244-253; wherein R1and / or R2is cleaved by TPA and comprises any one of SEQ ID NOS: 254-263; or wherein R1and / or R2is cleaved by UPA and comprises any one of SEQ ID NOS: 264-273. ^Attorney Docket No.10034-321WO1 15. The protease activity sensor of any one of claims 1-14, wherein R1and / or R2are each independently selected from IEFDSG (SEQ ID NO: 10), APAALRAA (SEQ ID NO: 11), AAN (SEQ ID NO: 12), ASGPAGPA (SEQ ID NO: 13), PAALRA (SEQ ID NO: 14), fPRSG (SEQ ID NO: 15), LVXXXSG (SEQ ID NO: 19), LVSPRSG (SEQ ID NO: 20), LVSFPSG (SEQ ID NO: 21), LVQNLSG (SEQ ID NO: 22), LVPRGSG (SEQ ID NO: 23), and PLGLAG (SEQ ID NO: 24).

16. The protease activity sensor of any one of claims 1-15, wherein R1and R2have different sequences that are cleaved by the same protease.

17. The protease activity sensor of any one of claims 1-15, wherein R1and R2have the same sequence.

18. A nanoparticle comprising a plurality of the protease activity sensors of any one of claims 1-17 anchored to a surface of said nanoparticle.

19. The nanoparticle of claim 18, wherein the nanoparticle comprises a plurality of thiol- reactive moieties on its surface; wherein X5is Ck; and wherein the thiol-reactive moieties are crosslinked to the cysteine in X5, thereby anchoring the plurality of protease activity sensors to the surface of the nanoparticle.

20. The nanoparticle of claim 19, wherein the plurality of thiol-reactive moieties comprise iodoacetyl, maleimide, or any combination thereof.

21. The nanoparticle of any one of claims 18-20, wherein the nanoparticle comprises from about 2 to about 200 protease activity sensors anchored on its surface.

22. The nanoparticle of any one of claims 18-21, wherein, upon cleavage of both R1and R2, the reporter molecule remains anchored to the surface of the nanoparticle; and wherein X3is a quencher or an acetyl group and X4is the reporter molecule.

23. The nanoparticle of any one of claims 18-21, wherein, upon cleavage of both R1and R2, the reporter molecule is released from the nanoparticle. ^Attorney Docket No.10034-321WO1 24. The nanoparticle of claim 23, wherein X3is the reporter molecule and X4is null or a quencher.

25. A method of detecting presence of a protease, the method comprising: a) providing the protease activity sensor of any one of claims 1-17 or the nanoparticle of any one of claims 18-24 to a sample or a subject, wherein R1and R2are protease cleavage sites for the same protease; and b) detecting presence or absence of the reporter molecule in the sample, the subject, or a sample taken from the subject, thereby detecting presence or absence of the protease; wherein presence of the reporter molecule indicates presence of the protease; and wherein absence of the reporter molecule indicates absence of the protease.

26. The method of claim 25, wherein step a) further comprises: i) providing the protease activity sensor to the subject; and ii) collecting a biological fluid sample from the subject; and wherein step b) further comprises detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the protease.

27. The method of claim 25, wherein step a) further comprises providing the nanoparticle to the subject, wherein upon cleavage of both R1and R2, the reporter molecule remains anchored to the surface of the nanoparticle; and wherein step b) further comprises imaging the subject to detect presence or absence of the reporter molecule, thereby locally detecting presence or absence of the protease.

28. The method of claim 25, wherein step a) further comprises: i) providing the nanoparticle to the subject, wherein, upon cleavage of both R1and R2, the reporter molecule is released from the nanoparticle; and ii) collecting a biological fluid sample from the subject; and wherein step b) further comprises detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the protease. ^Attorney Docket No.10034-321WO1 29. The method of any one of claims 25-28, wherein presence of protease indicates presence and / or severity of a disease or disorder; and wherein the method is used to determine if the subject has said disease or disorder and / or progression of said disease or disorder.

30. The method of any one of claims 25-29, wherein presence of the protease indicates an immune response to a disease or disorder; and wherein the method is used to determine responsiveness of said disease or disorder to a treatment.

31. The method of claim 30, wherein the treatment is an immunotherapy.

32. The method of any one of claims 30-31, wherein the disease or disorder is cancer.

33. The method of claim 32, wherein the subject has a tumor; and wherein step a) further comprises injecting the protease activity sensor or the nanoparticle into said tumor.

34. A method of detecting presence of two proteases, the method comprising: a) providing the protease activity sensor of any one of claims 1-17 or the nanoparticle of any one of claims 18-24 to a sample or a subject, wherein R1and R2are protease cleavage sites for different proteases; and b) detecting presence or absence of the reporter molecule in the sample, the subject, or a sample taken from the subject, thereby detecting presence or absence of the two proteases; wherein presence of the reporter molecule indicates presence of both of the two proteases; and wherein absence of the reporter molecule indicates absence of one or both of the two proteases.

35. The method of claim 34, wherein step a) further comprises: i) providing the protease activity sensor to the subject; and ii) collecting a biological fluid sample from the subject; and ^Attorney Docket No.10034-321WO1 wherein step b) further comprises detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the two proteases.

36. The method of claim 34, wherein step a) further comprises providing the nanoparticle to the subject, wherein upon cleavage of both R1and R2, the reporter molecule remains anchored to the surface of the nanoparticle; and wherein step b) further comprises imaging the subject to detect presence or absence of the reporter molecule, thereby locally detecting presence or absence of the two proteases.

37. The method of claim 34, wherein step a) further comprises: i) providing the nanoparticle to the subject, wherein, upon cleavage of both R1and R2, the reporter molecule is released from the nanoparticle; and ii) collecting a biological fluid sample from the subject; and wherein step b) further comprises detecting presence or absence of the reporter molecule in the biological fluid sample, thereby distally detecting presence or absence of the two proteases.

38. The method of any one of claims 34-37, wherein presence of both of the two proteases indicates presence and / or severity of a disease or disorder; and wherein the method is used to determine if the subject has said disease or disorder and / or progression of said disease or disorder.

39. The method of any one of claims 34-38, wherein presence of both of the two protease indicates an immune response to a disease or disorder; and wherein the method is used to determine responsiveness of said disease or disorder to a treatment.

40. The method of claim 39, wherein the treatment is an immunotherapy.

41. The method of any one of claims 39-40, wherein the disease or disorder is cancer.

42. The method of claim 41, wherein the subject has a tumor; and ^Attorney Docket No.10034-321WO1 wherein step a) further comprises injecting the protease activity sensor or the nanoparticle into said tumor. ^

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