Tandem molecular fluorescence reporter for detection of tumor infiltrating leukocytes

Abstract

This invention relates to compounds of formula (I): wherein, X ‑ A represents an anti-charged ion; and A represents an amino acid moiety that can be cleaved by leukocyte-associated enzymes, or its pharmaceutically acceptable salt or solvate.

Description

Technical Field

[0001] This invention relates to diagnostic molecules that can be used in vivo or in vitro to determine whether a subject has a specific disease or whether that disease makes the subject susceptible to a specific treatment. The invention also relates to methods for preparing said compounds and compounds for the aforementioned uses. Background Technology

[0002] The listing or discussion of previously disclosed documents in this specification should not necessarily be construed as an admission that such documents are part of the prior art or common general knowledge.

[0003] Cancer immunotherapy, which trains the immune system to eradicate cancer, has revolutionized the landscape of oncology. However, patient responses to the same immunotherapy often vary, and response rates to checkpoint blockade therapy remain low in many cancer types (20-40% of melanoma, renal cell carcinoma, and colorectal cancer). Tumor-infiltrating leukocytes (TILs) are known to be strongly associated with cancer progression and clinical endpoints in cancer patients. In particular, clinical data have revealed that high levels of M1 macrophages and cytotoxic T lymphocytes (CTLs) are associated with a positive prognosis (Fridman, W. He et al., Nat. Rev. Clin. Oncol. 2017, 14, 717-734), while an increased neutrophil-lymphocyte ratio predicts low survival in cancer patients (Gentles, A. Je et al., Nat. Med. 2015, 21, 938-945). To assess tumor-associated lymphoid tissue (TIL) within the tumor immune microenvironment (TIME) for patient stratification and treatment prediction before and during cancer immunotherapy, flow cytometry, histological staining, and mass spectrometry of biopsied tumor tissue have been used. However, single-point biopsies are invasive, static, carry a risk of metastasis, and only reveal regional information about the TIME; while peripheral blood analyses inevitably include biomarkers secreted from organs other than tumors, thus reducing the specificity for the TIME.

[0004] Molecular imaging offers a real-time and non-invasive approach for longitudinal monitoring of overall time-related leukocytes (TILs). However, existing imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), cannot accurately monitor tumor-associated lymphoid tissue (TILs) within TILs because they rely on antibody / ligand-conjugated contrast agents to target specific leukocytes, and their "always-on" signals are inevitably affected by non-specific retention in tissues outside the tumor. In contrast, activatable molecular optical reporters trigger their signals only against the intended biomarkers, thus exhibiting minimized background and quantitative signals related to the concentration and activity of the biomarkers. Recently, activatable molecular reporters have been developed for real-time leukocyte imaging; however, because their activation mechanisms are determined solely by leukocyte biomarkers, their signals can be non-specifically triggered by leukocytes in peripheral blood and inflamed tissues. Therefore, specific real-time non-invasive imaging of TILs remains a significant challenge.

[0005] Therefore, new molecular fluorescence reporters are needed for the detection of TILs. Summary of the Invention

[0006] The various aspects and implementation methods will now be discussed with reference to the following entries.

[0007] 1. Compounds of Formula I:

[0008]

[0009] in:

[0010] X - Represents an anti-charge ion; and

[0011] A represents an amino acid moiety that can be cleaved by enzymes associated with leukocytes, or its pharmaceutically acceptable salt or solvate.

[0012] 2. The compound or its salt or solvate as described in item 1, wherein A is selected from:

[0013]

[0014]

[0015] The attached points are represented by dashed lines.

[0016] 3. Use of a compound of formula I, or a salt or solvate thereof, as defined in entry 1 or 2, in the preparation of an imaging agent for the diagnosis of lesions or diseases in tissues and / or organs using near-infrared fluorescence.

[0017] 4. The use of a compound of formula I, or a salt or solvate thereof, as defined in entry 1 or 2, as an imaging agent for the diagnosis of symptoms or diseases in tissues and / or organs using near-infrared fluorescence.

[0018] 5. A compound for use according to item 3 or according to item 4, wherein the use is in vivo or in vitro.

[0019] 6. The compound for the use or purpose described in item 5, wherein in vivo imaging is for the purpose of visualizing the tumor immune microenvironment.

[0020] 7. A method for diagnosing a symptom or disease in a tissue and / or organ, the method comprising the steps of: administering to a subject requiring diagnosis a compound of formula I as defined in entry 1 or entry 2, or a salt or solvate thereof, and using near-infrared fluorescence to determine the presence or absence of a symptom or disease in the tissue and / or organ.

[0021] 8. A method for determining the susceptibility of tumor tissue to immunotherapy, the method comprising the steps of:

[0022] (i) subjecting tumor tissue to the required course of immunotherapy;

[0023] (ii) administering to the tumor tissue a compound of formula I as defined in entry 1 or entry 2, or a salt or solvation thereof, together with a reference report compound that is always open.

[0024] (iii) After a period of time, determine the ratio of near-infrared fluorescence obtained from the metabolite of the compound of formula I as defined in item 1 or 2 to that of the reference reporter compound that is always on; and

[0025] (iv) Determine the susceptibility of the tumor tissue to immunotherapy based on the ratio obtained in step (iii).

[0026] 9. The method according to item 8, wherein the method is performed in vivo.

[0027] 10. The method according to item 8, wherein the method is performed in vitro.

[0028] 11. The method according to any one of items 8 to 10, wherein the method further relates to a treatment plan for the subject based on the results obtained by the method.

[0029] 12. The method according to any one of items 8 to 11, wherein the always-on reference reporter compound is:

[0030] Attached Figure Description

[0031] Figure 1 illustrates the design and mechanism of tandemly activatable molecular reporter (TAMR) for specific molecular optical imaging of TILs. (a) Schematic diagram of targeted TILs in tumors, real-time, non-invasive, and specific imaging of TILs during cancer immunotherapy using TAMR, and the potential applications of TAMR in patient stratification and treatment efficacy prediction. (b) Including TAMR M1 TAMR CTL and TAMR NE The chemical structures of TAMRs and their activation processes in the presence of cancer and leukocyte biomarkers, as well as the chemical structure of the tumor penetration reference report, polyvinylpyrrolidone (PVP)-IR800. (c) Schematic diagram of TAMR monitoring of TILs. After TAMRs accumulate to TIME, their corresponding TAMR peptide substrates (R2 fragments) are cleaved by nitric oxide (NO) overexpressed by tumor-infiltrating M1 macrophages and activated caspase-1 (Cas-1), by granzyme B (GrB) overexpressed by tumor-infiltrating CTLs, and by NE overexpressed by tumor-infiltrating neutrophils. Subsequently, tumor biomarkers (APNs) can attack the exposed alanine (R1 fragment), leading to the activation of TAMR fluorescent signals.

[0032] Figure 2 illustrates the synthesis of TAMR. (a) Synthesis of the fluorescent signal transduction moiety (CyOH) and the tumor passive targeting moiety (PVP-acetylene): (i) resorcinol, K2CO3, acetonitrile (CH3CN), 55 °C, 6 h; (ii) isopropoxyethanol, 2,2'-azobisisobutyronitrile (AIBN), 60 °C, 4 h; and (iii) bromopropyne, tetrahydrofuran (THF), 25 °C, 24 h. (b) Synthesis of CyA: (i) p-aminobenzyl alcohol (PABA), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), dichloromethane (DCM), 25 °C, 6 h; (ii) PBr3, THF, 0 °C, 2 h; (iii) CyOH, N,N-diisopropylethylamine (DIPEA), CH3CN, 55 °C, 8 h; and (iv) piperidine, dimethylformamide (DMF), 25 °C, 5 min. (c) Synthesis of TASMR: (i) Ac-Y(tBu)VAD(OtBu)-OH, benzotriazole-tetramethylurea hexafluorophosphate (HBTU), hydroxybenzotriazole (HOBt), DIPEA, DMF, 25℃, 2h; (ii) trifluoroacetic acid (TFA) / H2O, 0℃, 40min; (iii) o-phenylenediamine (OPD), HBTU, HOBt, DIPEA, DMF, 25℃, 0.5h; (iv) Ac-IE(OtBu)FD(OtBu)-OH, HBTU, HOBt, DIPEA, DMF, 25℃, 2h; (v) TFA / H2O, 0℃, 40min; and (vi) MeOSuc-AAPV-OH, HBTU, HOBt, DIPEA, DIPEA, 25℃, 2h. (d) Synthesis of TAMR: (i) PVP-acetylenes, CuSO4·5H2O, sodium ascorbate, H2O / dimethyl sulfoxide (DMSO), 25℃, 12h.

[0033] Figure 3The pharmacokinetics of (a) polyethylene glycol (PEG)-Cy and (b) PVP-Cy in healthy mice following intravenous (iv) injection of two probes were depicted. Unlocked PEG-Cy and PVP-Cy were administered intravenously to healthy mice. [Cy] = 5 μmol / kg. The circulating half-lives of PVP-Cy and PEG-Cy were calculated to be 48 min and 11 min, respectively. (c) Real-time imaging of CT26 tumor-bearing mice after intravenous injection of PEG-Cy or PVP-Cy (5 μmol / kg). NIRF images were acquired at 720 nm with excitation at 675 nm using an IVIS spectral imaging system. (d) Quantification of NIRF signal at the tumor site as a function of time points after probe injection. The NIRF signal in the tumor region gradually increased, reaching its maximum at 6 h and 8 h after PVP-Cy and PEG-Cy injection, respectively. Error bars represent the standard deviation of three individual measurements.

[0034] Figure 4 The description depicts (a)TAMR in the presence of a single biomarker or a combination of biomarkers. M1 (b)TAMR CTL and (c)TAMR NE UV-Vis absorption spectra in the corresponding buffers at 37°C. Note that GrB (0.5 μg) was first activated with cathepsin C (0.2 μg) in MES buffer for 4 h. APN buffer: 50 mM tris, pH 7.0. For TAMR M1 Diethylamine nonanoate (50 μM) was used as the NO source. [TASMR] = 25 μM. All enzymatic experiments were incubated at 37 °C for 2 h and were independently repeated three times, with similar results.

[0035] Figure 5 depicts the in vitro characterization of the detection capability of TAMR. In the presence of a single biomarker or a combination of biomarkers, (a) TAMR M1 (b)TAMR CTL and (c)TAMR NE Fluorescence spectra obtained at 37°C for 2 hours in the corresponding buffer solution. [TAMR] = 25 μM. Excitation: 680 nm. (d–f) In vitro selectivity of TAMR. (d) TAMR M1 (e)TAMR CTL and (f)TAMR NEChanges in near-infrared fluorescence (NIRF) at 720 nm (n=3) after incubation at 37 °C with single or combined biomarkers in their respective buffers for 2 h. [TAMR] = 25 μM. (d) 1: Blank; 2: Urokinase plasminogen activator (uPA); 3: Nitroreductase (NTR); 4: Gamma-glutamyl transferase (GGT); 5: Fibroblast activator protein (FAP); 6: Caspase-3; 7: Cathepsin S (CTSS); 8: GrB; 9: NE; 10: CTSS & APN; 11: GrB & APN; 12: NE & APN; 13: DPPI; 14: NO & Cas-1; 15: APN; and 16: NO & Cas-1 & APN. (e) 1: Blank; 2: uPA; 3: NTR; 4: GGT; 5: FAP; 6: Caspase-3; 7: NO & Cas-1; 8: CTSS; 9: NE; 10: NO & Cas-1 & APN; 11: CTSS & APN; 12: NE & APN; 13: DPPI; 14: GrB; 15: APN; and 16: GrB & APN. (f) 1: Blank; 2: uPA; 3: NTR; 4: GGT; 5: FAP; 6: Caspase-3; 7: NO & Cas-1; 8: GrB; 9: CTSS; 10: NO & Cas-1 & APN; 11: GrB & APN; 12: CTSS & APN; 13: DPPI; 14: NE; 15: APN; and 16: NE & APN. 16 relative to other groups: TAMR M1 P < 0.0001, TAMR CTL P < 0.0001, TAMR NE P < 0.0001. Confocal imaging and quantification of TAMR in (g, h) cells: M1 macrophages, CD8 T cells, and neutrophils in the presence or absence of APN (0.5 μg) with TAMR M1 TAMR CTL and TAMR NE (g) confocal imaging and (h) corresponding mean fluorescence intensity after 2 hours of co-incubation. [TAMR] = 10 μM. All confocal imaging experiments were independently repeated three times. Data are expressed as mean ± SD and analyzed by a two-tailed Student's t-test.

[0036] Figure 6 The effects of single or combined biomarkers (50 μM diethylamine nonanoate, 1 U Cas-1, 0.5 μg APN, 0.5 μg GrB, 2.5 mU NE) on (a) TASMR were described. M1 (b) TASMR CTL and (c)TASMRNE UV-Vis absorption spectra and (d)TASMR in the corresponding buffer solutions at 37°C M1 (e)TASMR CTL and (f)TASMR NE Fluorescence spectra in the corresponding buffers at 37°C. The excitation wavelength of the fluorescence spectra was 680 nm. All enzymatic experiments were incubated at 37°C for 2 h and were independently repeated three times, with similar results. [TASMR] = 25 μM. In the presence of both tumor (APN) and M1 macrophage biomarkers (Cas-1 and NO), the fluorescence signal of TASMRM1 at 710 nm increased 28-fold. In the presence of both tumor (APN) and CTL biomarker (GrB), the fluorescence signal of TASMRCTL at 710 nm increased 15-fold. In the presence of both tumor (APN) and neutrophil biomarker (NE), TASMR NE The fluorescence signal at 710 nm increased by 10 times.

[0037] Figure 7 Depicting (a) TASMR M1 (b) TASMR CTL and (c)TASMR NE NIRF changes at 720 nm after incubation at 37 °C with single or combined biomarkers in their respective buffers for 2 h. (a) 1: blank; 2: uPA; 3: NTR; 4: GGT; 5: FAP; 6: caspase-3; 7: CTSS; 8: GrB; 9: NE; 10: CTSS & APN; 11: GrB & APN; 12: NE & APN; 13: DPPI; 14: NO & Cas-1; 15: APN; and 16: NO & Cas-1 & APN. (b) 1: Blank; 2: uPA; 3: NTR; 4: GGT; 5: FAP; 6: Caspase-3; 7: NO & Cas-1; 8: CTSS; 9: NE; 10: NO & Cas-1 & APN; 11: CTSS & APN; 12: NE & APN; 13: DPPI; 14: GrB; 15: APN; and 16: GrB & APN. (c) 1: Blank; 2: uPA; 3: NTR; 4: GGT; 5: FAP; 6: Caspase-3; 7: NO & Cas-1; 8: GrB; 9: CTSS; 10: NO & Cas-1 & APN; 11: GrB & APN; 12: CTSS & APN; 13: DPPI; 14: NE; 15: APN; and 16: NE & APN. [TASMR] = 25 μM. All enzymatic experiments were independently repeated three times, and the results were similar. TASMR showed negligible enhancement after incubation with other interfering enzymes.

[0038] Figure 8 The study described the effects of single or combined biomarkers (50 μM diethylamine nonanoate, 1 U Cas-1, 0.5 μg APN, 0.5 μg GrB, 2.5 mU NE) on (a) TASMR. M1 (b) TASMR CTL and (c)TASMR NE High-performance liquid chromatography (HPLC) traces were obtained at 37°C for 2 hours in the corresponding buffer solutions. HPLC analyses of pure CyA and CyOH were used for comparison. HPLC traces of all intermediate enzymatic products were obtained in triplicate independently, and the results were similar.

[0039] Figure 9 The description of (a) Cas-1 to TASMR M1 (b) GrB on TASMR CTL (c)NE to TASMR NE Nonlinear regression analysis of the cleavage rate (V) of (d)APN to CyA as a function of substrate concentration was performed. Different concentrations of TASMR and CyA were incubated with the corresponding enzymes at 37°C.

[0040] Figure 10 The process of using (a)TAMR to synthesize CD8 T cells, M1 macrophages, neutrophils, 4T1 and CT26 cells at final concentrations of 12.5 to 100 μg / mL was described. M1 (b)TAMR CTL and (c)TAMR NE Cell viability after 24 hours of treatment.

[0041] Figure 11 The effects of APN (0.5 μg) in the presence or absence on 3T3 cells (mouse embryonic fibroblast cell line) in TAMR were described. M1 TAMR CTL and TAMR NE Representative confocal fluorescence imaging after 2 hours of co-incubation. [TAMR] = 10 μM.

[0042] Figure 12 The M0 phenotype of RAW264.7 cells (mouse macrophages) in the presence or absence of APN (0.5 μg) was depicted in relation to TAMR. M1 TAMR CTL and TAMR NE Representative confocal fluorescence imaging after 2 hours of co-incubation. [TAMR] = 10 μM.

[0043] Figure 13The M2 phenotype of RAW264.7 cells with or without APN (0.5 μg) was depicted in relation to TAMR. M1 TAMR CTL and TAMR NE Representative confocal fluorescence imaging after 2 hours of co-incubation. [TAMR] = 10 μM.

[0044] Figure 14 The effects of bone marrow-derived dendritic cells (BMDCs) in the presence or absence of APN (0.5 μg) on ​​TAMR were depicted. M1 TAMR CTL and TAMR NE Representative confocal fluorescence imaging after 2 hours of co-incubation. [TAMR] = 10 μM.

[0045] Figure 15 The 4T1 and CT26 cells were depicted in relation to TAMR M1 TAMR CTL and TAMR NE Representative confocal fluorescence imaging after 2 hours of co-incubation. [TAMR] = 10 μM.

[0046] Figure 16 illustrates the superiority of TAMRs compared to single-lock activatable molecular reporters (AMRs). (a) Including AMRs M1 AMR CTL and AMR NE The chemical structures of AMRs, including those in inflammatory leukocytes and TILs, and their activated forms. (b, c) Comparison of TAMR and AMR in blood samples: (b) Experimental protocol for comparing TAMR and AMR in blood samples from mice treated with saline or lipopolysaccharide (LPS); and (c) NIRF signals of TAMR and AMR (10 μM) after 30 min incubation in blood samples from mice treated with saline or LPS (n=3). Black lines indicate the NIRF intensity of these reporters at the same concentration in phosphate-buffered saline (PBS). Saline-treated group vs. LPS-treated group: no significant difference (ns). (d–f) Comparison of TAMR and AMR in live mice: (d) Schematic diagram comparing TAMR and AMR in live mice with LPS-inflamed tissue in the left thigh muscle and a subcutaneous CT26 tumor in the right flank; and (e) quantification of NIRF signals and (f) NIRF images (n=3) of the reporter (0.1 mM, 10 μL) in the LPS-inflamed and CT26 tumor tissues 30 min after local injection. Black lines represent the background NIRF intensity of these reporters in the skin. TAMR in tumor tissue relative to LPS-inflamed tissue. M1 P < 0.0001, TAMRCTL P < 0.0001, TAMR NE P = 0.0001, AMR: ns. (g–j) Comparison of TAMR and AMR via urinalysis: (g) Schematic diagram of the fluorescent urinalysis procedure; (h) Renal clearance efficiency of TAMR and AMR in healthy mice 24 h after intravenous injection of these reporters (n = 3); and (i) NIRF images and (j) quantification of NIRF signals of the reporters in urine collected from healthy mice 12 h after TAMR or AMR injection. Black lines indicate NIRF intensities of these reporters at the same concentration in PBS (n = 3). TAMR M1 Compared to AMR M1 P = 0.0006; TAMR CTL Compared to AMR CTL P = 0.0052; TAMR NE Compared to AMR NE P = 0.0003. All NIRF images were captured using an IVIS spectral imaging system. Excitation: 675 nm, emission: 720 nm. Data are expressed as mean ± SD and analyzed using a two-tailed Student's t-test.

[0047] Figure 17 The absorbance intensities of TAMR or AMR (10 μM) at 680 nm in the supernatant of homogenized LPS-inflamed blood samples and saline-treated blood samples were depicted. LPS (5.0 mg / kg) was intraperitoneally injected into live mice, and LPS-inflamed blood samples were collected 4 h post-injection. TAMR and AMR (10 μM) were incubated with both LPS-inflamed blood samples and saline-treated blood samples for 30 min. The blood samples were then homogenized and centrifuged for 10 min. The supernatant was used for absorbance analysis.

[0048] Figure 18 depicts (a) NIRF images of the reporter in LPS-inflamed tissue and CT26 tumor tissue after local injection of TAMR or AMR. (b) Quantification of NIRF intensity in LPS-inflamed tissue and CT26 tumor tissue as a function of time points after reporter injection. CT26 tumor was first inoculated into the right flank 7 days later, followed by an injection of LPS (5 mg / kg) into the left thigh muscle. 24 h later, 10 μL of TAMR or AMR (0.1 mM) was locally injected into LPS-inflamed tissue and CT26 tumor for longitudinal NIRF imaging. NIRF images were acquired at 720 nm with excitation at 675 nm using an IVIS spectral imaging system. *P<0.05, **P<0.01, ****P<0.0001. All experiments were independently repeated three times. Statistical significance was determined by a two-tailed Student's t-test. ns: No significance, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0049] Figure 19 The study describes (a) the effects of TAMR in the presence or absence of an APN inhibitor (bestatin). CTL Real-time imaging of 4T1 and CT26 tumor-bearing mice after intravenous injection of TAMR (5 μmol / kg) and PVP-IR800 (3 μmol / kg). CTL Two hours prior, betadine (10 mg / mL, 10 μL) was injected intratumorally. NIRF images were acquired at 720 nm using an IVIS spectral imaging system under excitation at 675 nm. (b) R-NIRF at the tumor site. CTL The quantification was used as a function of the time point after the reporter injection (n=3).

[0050] Figure 20 The study depicted the effects of (a)TAMR in healthy mice following intravenous injection of the probe. M1 (b)AMR M1 (c)TAMR CTL (d)AMR CTL (e)TAMR NE and (f)AMR NE Renal clearance efficiency (n=3) was determined. Healthy mice were intravenously injected with TAMR (5 μmol / kg) or AMR (5 μmol / kg) and placed in metabolic cages. Urine samples were collected at 3, 9, and 24 h post-injection. Renal clearance of these reporters was determined by HPLC analysis of the urine samples.

[0051] Figure 21The UV-Vis absorption spectra of the probes in urine collected from healthy mice are depicted. The shaded rectangles represent the peak of the AMR in urine at 680 nm.

[0052] Figure 22 depicts in vivo specific real-time NIRF imaging of TIL in 4T1 tumor-bearing mice. (a) Schematic diagram of the TIL immunotherapy process and real-time imaging. (b) Real-time imaging of 4T1 tumor-bearing mice after intravenous injection of TAMR (5 μmol / kg) and PVP-IR800 (3 μmol / kg) following different treatments. NIRF images obtained at 675 nm excitation and 720 nm emission. (c) R-NIRF at the tumor site as a function of time points after reporter injection (n=3). aPD-L1 vs. saline, TAMR M1 P = 0.0013; TAMR CTL P = 0.0215. aPD-L1 / oxaliplatin (Oxa) relative to saline, TAMR M1 P = 0.0003; TAMR CTL P = 0.0043; TAMR NE P = 0.0048. (d) Colocalization of NIRF signals of TAMR and their corresponding TILs in 4T1 tumor sections after treatment with aPD-L1 / Oxa (n = 3). Venn plots show the mean cell number, and the bottom panel shows the percentage of single-positive and double-positive cells for TAMR and their corresponding TILs. (e) In mouse tumors treated with different methods, the ratio of F4 / 80 + Cell-gated iNOS + Cas-1 + Cells, GrB gated by CD8+ cells + Cells and CD11b + Cell-gated Ly-6G + NE + Representative flow cytometry images of cells (n=3). (f) Correlation between TIL biomarkers and TAMR R-NIRF in tumors of 4T1 tumor-bearing mice treated with different methods, using a simple linear regression model. 95% confidence intervals were obtained through two-sided Student's t-test analysis. (g) Dynamic overview of TIME cells treated with different methods, and the corresponding TIL population percentage on CD45. + A schematic diagram of cell proportions. Data are expressed as mean ± SD and analyzed using a two-tailed Student's t-test.

[0053] Figure 23Real-time imaging of the reference reporter PVP-IR800 was depicted. (a) Real-time imaging of mice after intravenous injection of TAMR and their reference reporter (PVP-IR800) into 4T1 tumor-bearing mice treated with different methods. 4T1 tumor-bearing mice treated with different methods were intravenously injected with TAMR (5 μmol / kg) and monitored for real-time NIRF imaging for 48 h. NIRF images of PVP-IR800 (n=3) were obtained at 745 nm under 800 nm excitation using an IVIS spectral imaging system. (b) Quantification of the NIRF intensity of PVP-IR800 at the tumor site as a function of time points after reporter injection.

[0054] Figure 24 Ex vivo NIRF images (TAMR) of 4T1 tumor-bearing mice were depicted. M1 (a) In TAMR M1 (a) Ex vivo NIRF images of major organs (including heart, liver, spleen, lung, and kidney) and tumors in 4T1 tumor-bearing mice at a time point 72 h after injection of (5 μmol / kg) and PVP-IR800 (3 μmol / kg). (b) R-NIRF in resected tumors. M1 Quantitative analysis. Immunotherapy group vs. saline group: ***P<0.001, ****P<0.0001. (c) TAMR in major organs by HPLC. M1 Quantitative analysis was performed. (In TAMR) M1 Mice were dissected 72 h after injection of (5 μmol / kg) PBS. Major organs were homogenized in PBS and centrifuged to remove insoluble components. The supernatant containing the extracted reporter was analyzed by HPLC. Statistical significance was determined using a two-tailed Student's t-test. ns: no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0055] Figure 25 Ex vivo NIRF images (TAMR) of 4T1 tumor-bearing mice were depicted. CTL (a) In TAMR CTL (a) Ex vivo NIRF images of major organs (including heart, liver, spleen, lung, and kidney) and tumors in 4T1 tumor-bearing mice at a time point 72 h after injection of (5 μmol / kg) and PVP-IR800 (3 μmol / kg). (b) R-NIRF in resected tumors. CTL Quantitative analysis. Immunotherapy group vs. saline group: **P<0.01, ***P<0.001, ****P<0.0001. (c) TAMR in major organs analyzed by HPLC. CTL Quantitative analysis was performed. (In TAMR) CTLMice were dissected 72 h after injection of (5 μmol / kg) PBS. Major organs were homogenized in PBS and centrifuged to remove insoluble components. The supernatant containing the extracted reporter was analyzed by HPLC. Statistical significance was determined using a two-tailed Student's t-test. ns: no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0056] Figure 26 Ex vivo NIRF images (TAMR) of 4T1 tumor-bearing mice were depicted. NE (a) In TAMR NE (a) Ex vivo NIRF images of major organs (including heart, liver, spleen, lung, and kidney) and tumors in 4T1 tumor-bearing mice at a time point 72 h after injection of (5 μmol / kg) and PVP-IR800 (3 μmol / kg). (b) R-NIRF in resected tumors. NE Quantitative analysis. Immunotherapy group vs. saline group: *P<0.05, ****P<0.0001. (c) TAMR in major organs by HPLC. NE Quantitative analysis was performed. (In TAMR) NE Mice were dissected 72 h after injection of (5 μmol / kg) PBS. Major organs were homogenized in PBS and centrifuged to remove insoluble components. The supernatant containing the extracted reporter was analyzed by HPLC. Statistical significance was determined using a two-tailed Student's t-test. ns: no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0057] Figure 27 Representative immunofluorescence images of 4T1 tumor sections from mice treated with immunotherapy 24 h after intravenous injection of TAMRM1 (5 μmol / kg) were depicted. Images were acquired on an LSM800 (Zeiss) with a light source of 405 nm laser for the blue channel, 488 nm laser for the green channel, and 640 nm laser for the red channel. Blue fluorescence originated from cell nuclei stained with 4',6-diamidinyl-2-phenylindole (DAPI), green fluorescence from macrophages stained with F4 / 80 antibody labeled with FITC, and red fluorescence from activated TAMRM1. M1 (b) TAMR in 4T1 tumor sections after treatment with Oxa and aPD-L1 M1 With F4 / 80 + Colocalization of NIRF signals in cells. The Venn diagram shows the mean cell number, and the lower right corner represents TAMR. M1 The percentage of single-positive and double-positive cells and biomarkers.

[0058] Figure 28 The description of (a) TAMR injection via IV is as follows CTL Representative immunofluorescence images of 4T1 tumor sections from mice treated with immunotherapy (5 μmol / kg) for 24 h. Images were acquired on an LSM800 (Zeiss) with a light source of 405 nm laser for the blue channel, 488 nm laser for the green channel, and 640 nm laser for the red channel. (b) TAMR images of 4T1 tumor sections after treatment with Oxa and aPD-L1. CTL With CD8 + Colocalization of NIRF signals in cells. The Venn diagram shows the mean cell number, and the lower right corner represents TAMR. CTL The percentage of single-positive and double-positive cells and biomarkers.

[0059] Figure 29 The description of (a) TAMR injection via IV is as follows NE Representative immunofluorescence images of 4T1 tumor sections from mice treated with immunotherapy (5 μmol / kg) for 24 h. Images were acquired on an LSM800 (Zeiss) with a light source of 405 nm laser for the blue channel, 488 nm laser for the green channel, and 640 nm laser for the red channel. (b) TAMR images of 4T1 tumor sections after treatment with Oxa and aPD-L1. NE with NE + Colocalization of NIRF signals in cells. The Venn diagram shows the mean cell number, and the lower right corner represents TAMR. NE The percentage of single-positive and double-positive cells and biomarkers.

[0060] Figure 30 depicts flow cytometry analysis of M1 macrophages in tumor-draining lymph nodes and blood samples from 4T1 tumor-bearing mice treated with different methods on day 8. (a) Gating strategies used to analyze M1 macrophages in tumor, lymph node, and blood samples. (b) iNOS in tumor-draining lymph nodes. + Cells and CD11b + Cellular gated Cas-1 + iNOS + Representative flow cytometry images of cells. (c) iNOS in blood samples. + Cells and CD11b + Cellular gated Cas-1 + iNOS + Representative flow cytometry image of the cell.

[0061] Figure 31 depicts flow cytometry analysis of CTLs in tumor-draining lymph nodes and blood samples from 4T1 tumor-bearing mice treated with different methods on day 8. (a) Gating strategies used to analyze CTLs in tumor, lymph node, and blood samples. (b) CD8+ in tumor-draining lymph nodes. + Cellular gated CD8 + GrB + Representative flow cytometry images of cells. (c) CD8 in blood samples. + Cellular gated CD8 + GrB + Representative flow cytometry image of the cell.

[0062] Figure 32 Flow cytometry analysis of neutrophils in tumor and blood samples from 4T1 tumor-bearing mice treated with different methods on day 8 is depicted. (a) Gating strategies used to analyze neutrophils in tumor and blood samples. (b) CD45 in tumors. + Cellular gated CD11b + Ly-6G + Representative flow cytometry images of cells. (c) CD45 in blood samples. + Cellular gated CD11b + Ly-6G + Representative flow cytometry image of the cell.

[0063] Figure 33 The correlation between the tumor-infiltrating lymphocyte (TIL) population and the relative risk factor (R-NIRF) of TAMR in tumor regions of 4T1 tumor-bearing mice treated with different methods was depicted. R and P values ​​were derived using a simple linear regression model. The grey error bands indicate the 95% confidence intervals of the fitted lines analyzed by a two-sided Student's t-test.

[0064] Figure 34 depicts in vivo specific real-time NIRF imaging of TIL in CT26 tumor-bearing mice. (a) Schematic diagram of the TIL immunotherapy process and real-time imaging. (b) Real-time imaging of CT26 tumor-bearing mice after intravenous injection of TAMR (5 μmol / kg) and PVP-IR800 (3 μmol / kg) following different treatments. NIRF images obtained at 675 nm excitation and 720 nm emission. (c) R-NIRF at the tumor site as a function of time points after reporter injection (n=3). aPD-L1 relative to saline, TAMR CTL P < 0.0001; TAMR NE P < 0.0001. aCD47 relative to saline, TAMR M1 P = 0.0227; TAMR CTL P = 0.015; TAMR NEP = 0.0002. aPD-L1 / aCD47 relative to saline, TAMR M1 P = 0.0002; TAMR CTL P < 0.0001; TAMR NE P < 0.0001. (d) Colocalization of NIRF signals of TAMR and their corresponding TILs in CT26 tumor sections treated with aPD-L1 / aCD47 (n = 3). Venn plots show the mean cell number, and the lower plot shows the percentage of single-positive and double-positive cells for TAMR and their corresponding TILs. (e) In tumors of small CT26-bearing mice treated with different methods, the ratio of F4 / 80 + Cell-gated iNOS + Cas-1 + Cells, GrB gated by CD8+ cells + Cells and CD11b + Cell-gated Ly-6G + NE + Representative flow cytometry images of cells (n=3). (f) Correlation between TIL biomarkers and TAMR R-NIRF in tumors of CT26 tumor-bearing mice treated with different methods, using a simple linear regression model. 95% confidence intervals were obtained through two-sided Student's t-test analysis. (g) Dynamic overview of TIME cells treated with different methods, and the corresponding TIL population percentage on CD45. + A schematic diagram of cell proportions. Data are expressed as mean ± SD and analyzed using a two-tailed Student's t-test.

[0065] Figure 35 Real-time imaging of the reference reporter PVP-IR800 was depicted. (a) Real-time imaging of mice after intravenous injection of TAMR and their reference reporter (PVP-IR800) into CT26 tumor-bearing mice treated with different methods. CT26 tumor-bearing mice were intravenously injected with TAMR (5 μmol / kg) and PVP-IR800 (3 μmol / kg) and monitored for real-time NIRF imaging for 48 h (n=3). NIRF images of PVP-IR800 were acquired at 745 nm with excitation at 800 nm using an IVIS spectral imaging system. (b) Quantification of NIRF intensity of PVP-IR800 at the tumor site as a function of time points after reporter injection.

[0066] Figure 36 Ex vivo NIRF images (TAMR) of CT26 tumor-bearing mice were depicted. M1 (a) In TAMR M1(a) Ex vivo NIRF images of major organs (including heart, liver, spleen, lung, and kidney) and tumors in CT26 tumor-bearing mice 72 h after injection of (5 μmol / kg) and PVP-IR800 (3 μmol / kg). (b) R-NIRF in resected tumors. M1 Quantitative analysis. ***P<0.001, ****P<0.0001. (c) TAMR analysis of major organs by HPLC. M1 Quantitative analysis was performed. (In TAMR) M1 Mice were dissected 72 h after injection of (5 μmol / kg) PBS. Major organs were homogenized in PBS and centrifuged to remove insoluble components. The supernatant containing the extracted reporter was analyzed by HPLC. Statistical significance was determined using a two-tailed Student's t-test. ns: no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0067] Figure 37 Ex vivo NIRF images (TAMR) of CT26 tumor-bearing mice were depicted. CTL (a) In TAMR CTL (a) Ex vivo NIRF images of major organs (including heart, liver, spleen, lung, and kidney) and tumors in CT26 tumor-bearing mice 72 h after injection of (5 μmol / kg) and PVP-IR800 (3 μmol / kg). (b) R-NIRF in resected tumors. CTL Quantitative analysis. ***P<0.001, ****P<0.0001. (c) TAMR analysis of major organs by HPLC. CTL Quantitative analysis was performed. (In TAMR) CTL Mice were dissected 72 h after injection of (5 μmol / kg) PBS. Major organs were homogenized in PBS and centrifuged to remove insoluble components. The supernatant containing the extracted reporter was analyzed by HPLC. Statistical significance was determined using a two-tailed Student's t-test. ns: no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0068] Figure 38 Ex vivo NIRF images (TAMR) of CT26 tumor-bearing mice were depicted. NE (a) In TAMR NE (a) Ex vivo NIRF images of major organs (including heart, liver, spleen, lung, and kidney) and tumors in CT26 tumor-bearing mice 72 h after injection of (5 μmol / kg) and PVP-IR800 (3 μmol / kg). (b) R-NIRF in resected tumors. NEQuantitative analysis. ***P<0.001, ****P<0.0001. (c) TAMR analysis of major organs by HPLC. NE Quantitative analysis was performed. (In TAMR) NE Mice were dissected 72 h after injection of (5 μmol / kg) PBS. Major organs were homogenized in PBS and centrifuged to remove insoluble components. The supernatant containing the extracted reporter was analyzed by HPLC. Statistical significance was determined using a two-tailed Student's t-test. ns: no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0069] Figure 39 The description of (a) TAMR injection via IV is as follows M1 Representative immunofluorescence images of CT26 tumor sections from mice treated with immunotherapy (5 μmol / kg) for 24 h. Images were acquired on an LSM800 (Zeiss) with a light source of 405 nm laser for the blue channel, 488 nm laser for the green channel, and 640 nm laser for the red channel. (b) TAMR in CT26 tumor sections after treatment with aPD-L1 and aCD47. M1 With F4 / 80 + Colocalization of NIRF signals in cells. The Venn diagram shows the mean cell number, and the lower right corner represents TAMR. M1 The percentage of single-positive and double-positive cells and biomarkers.

[0070] Figure 40 The description of (a) TAMR injection via IV is as follows CTL Representative immunofluorescence images of CT26 tumor sections from mice treated with immunotherapy (5 μmol / kg) for 24 h. Images were acquired on an LSM800 (Zeiss) with a light source of 405 nm laser for the blue channel, 488 nm laser for the green channel, and 640 nm laser for the red channel. (b) TAMR in CT26 tumor sections after treatment with aPD-L1 and aCD47. CTL With CD8 + Colocalization of NIRF signals in cells. The Venn diagram shows the mean cell number, and the lower right corner represents TAMR. CTL The percentage of single-positive and double-positive cells and biomarkers.

[0071] Figure 41 The description of (a) TAMR injection via IV is as follows NERepresentative immunofluorescence images of CT26 tumor sections from mice treated with immunotherapy (5 μmol / kg) for 24 h. Images were acquired on an LSM800 (Zeiss) with a light source of 405 nm laser for the blue channel, 488 nm laser for the green channel, and 640 nm laser for the red channel. (b) TAMR in CT26 tumor sections after treatment with aPD-L1 and aCD47. NE and NE + Colocalization of NIRF signals in cells. The Venn diagram shows the mean cell number, and the lower right corner represents TAMR. NE The percentage of single-positive and double-positive cells and biomarkers.

[0072] Figure 42 depicts flow cytometry analysis of M1 macrophages in tumor-draining lymph nodes and blood samples from CT26 tumor-bearing mice treated with different methods on day 8. (a) Gating strategies used to analyze M1 macrophages in tumor, lymph node, and blood samples. (b) iNOS in tumor-draining lymph nodes. + Cells and CD11b + Cellular gated Cas-1 + iNOS + Representative flow cytometry images of cells. (c) iNOS in blood samples. + Cells and CD11b + Cellular gated Cas-1 + iNOS + Representative flow cytometry image of the cell.

[0073] Figure 43 depicts flow cytometry analysis of CTLs in tumor-draining lymph nodes and blood samples from CT26 tumor-bearing mice treated with different methods on day 8. (a) Gating strategies used to analyze CTLs in tumor, lymph node, and blood samples. (b) CD8+ in tumor-draining lymph nodes. + Cellular gated CD8 + GrB + Representative flow cytometry images of cells. (c) CD8 in blood samples. + Cellular gated CD8 + GrB + Representative flow cytometry image of the cell.

[0074] Figure 44 Flow cytometry analysis of neutrophils in tumor and blood samples from CT26 tumor-bearing mice treated with different methods on day 8 is depicted. (a) Gating strategies used to analyze neutrophils in tumor and blood samples. (b) CD45 in tumors. + Cellular gated CD11b+Ly-6G +Representative flow cytometry images of cells. (c) CD45 in blood samples. + Cellular gated CD11b + Ly-6G + Representative flow cytometry image of the cell.

[0075] Figure 45 The correlation between the tumor-infiltrating lymphocyte (TIL) population and the relative risk factor (R-NIRF) of TAMR in tumor regions of CT26 tumor-bearing mice treated with different methods was depicted. R and P values ​​were derived using a simple linear regression model. The gray error bands indicate the 95% confidence intervals of the fitted lines analyzed by a two-sided Student's t-test.

[0076] Figure 46 illustrates the prediction of cancer immunotherapy using TAMR. Schematic diagrams of the therapeutic mechanisms in (a) 4T1 tumor-bearing mice and (b) CT26 tumor-bearing mice. (c) Tumor growth curves (n=6) and (d) Overall survival curves (n=6) for 4T1 tumor-bearing mice. For tumor growth curves, aPD-L1 relative to saline: P=0.0005; Oxa relative to saline: P<0.0001; aPD-L1 / Oxa relative to saline: P<0.0001. For survival curves, aPD-L1 / Oxa relative to saline: P=0.0008. (e) Tumor growth curves (n=6) and (f) Overall survival curves (n=6) for CT26 tumor-bearing mice. For tumor growth curves, aPD-L1 relative to saline: P < 0.0001; aCD47 relative to saline: P = 0.0002; aPD-L1 / aCD47 relative to saline: P < 0.0001. For survival curves, aPD-L1 / aCD47 relative to saline: P = 0.0005. (g) Urinary R-NIRF of TAMR in tumor-bearing mice treated with different methods (n = 3). For 4T1 tumor-bearing mice: aPD-L1 relative to saline, TAMR M1 P = 0.0114; TAMR CTL P = 0.0123; TAMR NE ns. Oxa relative to saline: TAMR M1 P = 0.0047; TAMR CTL :ns;TAMR NE P = 0.0018. aPD-L1 / Oxa relative to saline: TAMR M1 P = 0.0006; TAMR CTL P = 0.0006; TAMR NE P = 0.0001. For CT26 tumor-bearing mice: aPD-L1 relative to saline, TAMR M1 P = 0.0002; TAMR CTLP = 0.0186; TAMR NE P = 0.0003. aCD47 relative to saline: TAMR M1 P < 0.0001; TAMR CTL :ns;TAMR NE P = 0.0002. aPD-L1 / aCD47 relative to saline: TAMR M1 P < 0.0001; TAMR CTL ::P=0.0003;TAMR NE P < 0.0001. (h) Correlation between urinary R-NIRF and relative tumor volume in TAMR of tumor-bearing mice with different treatments, using a simple linear regression model. 95% confidence intervals were obtained using a two-sided Student's t-test. (i) Principal component analysis (PCA) of R-NIRF in TAMR of untreated CT26 and 4T1 tumor-bearing mice. (j) Using R-NIRF M1 (Area under the curve (AUC) = 0.92, 95% CI = 0.757–1.000), R-NIRF CTL (AUC=0.98, 95%CI=0.917-1.000), R-NIRF NE (AUC = 0.53, 95% CI = 0.182–0.874) and derived from TAMR M1 TAMR CTL and TAMR NE The receiver operating characteristic (ROC) curves of TAMR were used to distinguish between untreated 4T1 and CT26 tumors by logistic regression of multiple TAMRs (AUC = 1.00, 95% CI = 1.000–1.000). (k) PCA of R-NIRF in 4T1 and CT26 tumor-bearing mice treated with aPD-L1. (j) Using R-NIRF M1 (AUC=0.81, 95%CI=0.616-1.000), R-NIRF CTL (AUC=0.93, 95%CI=0.781-1.000), R-NIRF NE (AUC = 0.96, 95% CI = 0.880-1.000) and derived from TAMR M1 TAMR CTL and TAMR NEThe logistic regression of R-NIRF on multiple TAMRs (AUC = 1.00, 95% CI = 1.000–1.000) was used to distinguish TAMR (ROC) curves between 4T1 and CT26 tumors treated with aPD-L1. (m) PCA of TAMR in tumor-bearing mice treated with different methods. (n) Whole-slide imaging analysis of TAMR distribution and (o) Quantification of TAMR fluorescence spots in tumor sections of mice treated with different methods at 24 h post-TAMR injection (n = 3). Scale bar = 500 μm. Data are expressed as mean ± SD and analyzed by a two-tailed Student's t-test.

[0077] Figure 47 Tumor control rate and body weight in mice were depicted. Tumor control rate at the end of treatment in (a) 4T1 tumor-bearing mice and (b) CT26 tumor-bearing mice. ****P<0.0001. (c) Body weight of 4T1 tumor-bearing mice after intraperitoneal injection of saline, Oxa (6 mg / kg), aPD-L1 (10 mg / kg), and aPD-L1 / Oxa on days 0, 2, and 4. (d) Body weight of CT26 tumor-bearing mice after intraperitoneal injection of saline, aCD47 (6 mg / kg), aPD-L1 (10 mg / kg), and aPD-L1 / aCD47 on days 0, 2, and 4. Statistical significance was determined by a two-tailed Student's t-test. ns: no significance, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0078] Figure 48 NIRF imaging of TAMR in urine of tumor-bearing mice treated with different methods was performed (n=3). Tumor-bearing mice treated with different methods were intravenously injected with PVP-IR800 (3 μmol / kg) and TAMR (5 μmol / kg) and placed in metabolic cages. Urine samples were collected at 12 time points post-injection and imaged using an IVIS spectral imaging system with an exposure time of 0.1 s, where excitation at 675 nm and emission at 720 nm were used to monitor activated CyOH.

[0079] Figure 49 The correlation between urinary R-NIRF and in vivo R-NIRF of TAMR in tumor regions of tumor-bearing mice treated with different methods was depicted. R and P values ​​were derived using a simple linear regression model. The grey error bands indicate the 95% confidence intervals of the fitted lines analyzed by a two-sided Student's t-test.

[0080] Figure 50 The correlation between urinary nitric acid (TIL) and tachycardia-reactive protein (TAMR) in tumor-bearing mice treated with different methods was depicted. R and P values ​​were derived using a simple linear regression model. The grey error bands indicate the 95% confidence intervals of the fitted lines analyzed by a two-sided Student's t-test.

[0081] Figure 51 The correlation between R-NIRF of real-time imaging TAMR in the tumor region and tumor progression, represented by the relative tumor volume in tumor-bearing mice after different treatments, was depicted. R and P values ​​were derived using a simple linear regression model. The grey error bands show the 95% confidence intervals of the fitted line analyzed by a two-sided Student's t-test.

[0082] Figure 52 (a) ROC analysis is depicted, which shows the results using TAMR. M1 R-NIRF (AUC=0.90, 95%CI=0.738-1.00), TAMR CTL R-NIRF (AUC=0.89, 95% CI=0.742-1.00), TAMR NE The specificity and sensitivity of R-NIRF (AUC = 0.84, 95% CI = 0.626–1.00) and R-NIRF of combinations of multiple TAMRs (AUC = 0.98, 95% CI = 0.920–1.00) were used to distinguish between untreated and all-treated 4T1 tumor-bearing mice. (b) ROC analysis, which showed the specificity and sensitivity of R-NIRF using TAMRs. M1 R-NIRF (AUC=0.83, 95%CI=0.683-0.971), TAMR CTL R-NIRF (AUC=0.88, 95%CI=0.731-1.00), TAMR NE The specificity and sensitivity of the R-NIRF (AUC = 0.97, 95% CI = 0.914–1.00) and the R-NIRF of a combination of multiple TAMRs (AUC = 1.00, 95% CI = 1.00–1.00) were used to distinguish between untreated and fully treated CT26 tumor-bearing mice. Dashed lines indicate randomized diagnostic tools with an AUC of 0.5.

[0083] Figure 53 The fluorescence intensity distribution of TAMR in whole-slice tumor sections was analyzed as a function of the radius from the center of the section. Quantitative data were obtained through image J analysis. Detailed Implementation

[0084] In a first aspect of the invention, a compound of formula I is provided:

[0085]

[0086] in:

[0087] X - Represents an anti-charge ion; and

[0088] A represents an amino acid moiety that can be cleaved by enzymes associated with leukocytes, or its pharmaceutically acceptable salt or solvate.

[0089] X - It can be any suitable anti-charged ion, such as a halide anti-charged ion (e.g., Cl-). - ,Br - or I - ).

[0090] In the embodiments described herein, the word "comprising" can be interpreted as requiring the mentioned features but not limiting the presence of other features. Alternatively, the word "comprising" can also refer to situations where only the listed components / features are intended to be present (e.g., the word "comprising" can be replaced by the phrases "consisting of" or "substantially consisting of"). It is explicitly contemplated that both the broader and narrower interpretations are applicable to all aspects and embodiments of the invention. In other words, the word "comprising" and its synonyms can be replaced by the phrases "consisting of" or "substantially consisting of" or their synonyms, and vice versa.

[0091] The phrase “consistent with…” and its pseudonym can be interpreted in this text as referring to materials that may contain small amounts of impurities. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0092] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, references to “composition” include mixtures of two or more such compositions, etc.

[0093] References to compounds of Formula I in this document (in any aspect or embodiment of the invention) include references to such compounds themselves, tautomers of such compounds, and pharmaceutically acceptable salts or solvates or pharmaceutically functional derivatives of such compounds.

[0094] Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts can be formed by conventional means, for example by reacting a compound of formula I in its free acid or free base form with an equivalent or greater equivalent of a suitable acid or base, optionally in a solvent or medium in which the salt is insoluble, followed by removal of the solvent or medium using standard techniques (e.g., by lyophilization or filtration in a vacuum). Salts can also be prepared by exchanging the anti-charged ion of a compound of formula I in its salt form with another anti-charged ion, for example using a suitable ion exchange resin.

[0095] Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, as well as salts derived from metals, such as sodium, magnesium, or preferably potassium and calcium.

[0096] Examples of acid addition salts include those formed from: acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, arylsulfonic acids (e.g., benzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, and p-toluenesulfonic acid), ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+)camphoric acid, camphor-sulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactopyric acid, gentian acid, glucoheponic acid, gluconic acid (e.g., D-gluconic acid), glucuronic acid ( For example, D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxomylglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxyethanesulfonic acid, lactic acid (e.g., (+)-L-lactic acid and (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid (e.g., (-)-L-malic acid), malonic acid, (±)-DL-mandelic acid, metaphosphoric acid, methanesulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid (e.g., (+)-L-tartaric acid), thiocyanate, undecenoic acid, and valeric acid.

[0097] Specific examples of salts are derived from the following: mineral acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid; organic acids, such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acid; and metals, such as sodium, magnesium, or preferably potassium and calcium.

[0098] As mentioned above, Formula I also encompasses any solvates of the compound and their salts. Preferred solvates are those formed by incorporating molecules of a non-toxic, pharmaceutically acceptable solvent (hereinafter referred to as a solvating solvent) into the solid-state structure (e.g., crystal structure) of the compound of the present invention. Examples of such solvents include water, alcohols (such as ethanol, isopropanol, and butanol), and dimethyl sulfoxide. Solvates can be prepared by recrystallizing the compound of the present invention with a solvent or a mixture of solvents containing a solvating solvent. In any given case, the formation of a solvate can be determined by analyzing the crystals of the compound using well-known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC), and X-ray crystallography.

[0099] The solvate can be stoichiometric or non-stoichiometric. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates, and dihydrates.

[0100] For a more detailed discussion of solvates and the methods used to manufacture and characterize them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc. of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0101] Compounds of Formula I may contain double bonds, and therefore may exist as E (opposite-sided) and Z (same-sided) geometric isomers for each individual double bond. All such isomers and mixtures thereof are included within the scope of this invention.

[0102] Compounds of Formula I can exist as regioisomers and can also exhibit tautomerism. All tautomer forms and mixtures thereof are included within the scope of this invention.

[0103] Compounds of Formula I may contain one or more asymmetric carbon atoms and may therefore exhibit optical and / or diastereomeric isomerism. Diastereomers can be separated using conventional techniques such as chromatography or fractional crystallization. Various stereoisomers can be separated by using conventional techniques such as fractional crystallization or HPLC to separate racemic or other mixtures of the compound. Alternatively, desired optical isomers can be prepared by: reacting a suitable optically active starting material under conditions that do not cause racemization or epimerization (i.e., a 'chiral pool' method); reacting a suitable starting material with a 'chiral auxiliary,' which can then be removed at a suitable stage; derivatization (i.e., resolution, including dynamic resolution), for example with an isochiral acid, followed by separation of the diastereomeric derivative by conventional means such as chromatography; or reaction with all suitable chiral reagents or chiral catalysts under conditions known to those skilled in the art. All stereoisomers and mixtures thereof are included within the scope of this invention.

[0104] In the implementation of the first aspect, A may be selected from:

[0105]

[0106]

[0107] The attached points are represented by dashed lines.

[0108] References for (a): J. Leukoc. Biol. 2016, 100, 961; Nat. Med. 2016, 22, 64; J. Biol. Chem. 1997, 272, 9677; and Adv. Mater. 2020, 32, 2000648. References for (b): J. Biol. Chem. 2007, 282, 4545; and Cancer Res. 2017, 77, 2318. References for (c): J. Biol. Chem. 1979, 254, 4027.

[0109] Compounds of Formula I can be used to help determine whether a subject has a specific disease. Therefore, in another aspect of the invention, the following is provided:

[0110] (aa) Use of a compound of formula I, as defined herein, or its salt or solvate, in the preparation of an imaging agent for the diagnosis of symptoms or diseases in tissues and / or organs using near-infrared fluorescence;

[0111] (ab) The use of compounds of formula I, or their salts or solvates, as defined herein, as imaging agents for the diagnosis of conditions or diseases in tissues and / or organs using near-infrared fluorescence; and

[0112] (ac) A method for diagnosing a symptom or disease in a tissue and / or organ, the method comprising the steps of administering a compound of formula I, as defined herein, or a salt or solvate thereof, to a subject requiring diagnosis, and using near-infrared fluorescence to determine the presence or absence of a symptom or disease in the tissue and / or organ.

[0113] The use of (aa) or (ab) of the compound can be performed in vivo or in vitro. In a particular embodiment, in vivo imaging may be used to visualize the tumor immune microenvironment.

[0114] Compounds of Formula I can also be used to determine whether a specific tumor tissue is susceptible to immunotherapy. Therefore, in another aspect of the invention, a method for determining the susceptibility of tumor tissue to immunotherapy is provided, the method comprising the steps of:

[0115] (i) subjecting tumor tissue to the required course of immunotherapy;

[0116] (ii) administering to tumor tissue a compound of formula I as defined herein, or a salt or solvate thereof, together with a reference report compound that is always open;

[0117] (iii) After a period of time, determine the ratio of near-infrared fluorescence obtained from the metabolite of the compound of formula I as defined herein to that of the reference reporter compound that was always on; and

[0118] (iv) Determine the susceptibility of tumor tissue to immunotherapy based on the ratio obtained in step (iii).

[0119] This method can be performed in vivo or in vitro.

[0120] The methods described above, whether performed in vitro or in vitro, can determine whether a specific tissue is treatable with immunotherapy. Therefore, if it is revealed that a tumor is susceptible to a planned treatment, the practitioner can treat the tumor. Thus, in some embodiments, the method may additionally involve formulating a treatment plan for a subject based on the results obtained through this method.

[0121] This document may use any suitable always-on reference reporter compound. For example, the reference reporter compound could be:

[0122]

[0123] To avoid any doubt, in the context of this invention, the term "treatment" includes references to therapeutic or palliative treatment for patients who require such treatment, as well as to preventive treatment and / or diagnosis for patients susceptible to relevant disease states.

[0124] The terms "patient" and "patients" include references to mammalian patients (e.g., humans). As used herein, the terms "subject" or "patient" are recognized in the art and are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cows, horses, goats, sheep, pigs, camels, and most preferably humans. In some embodiments, a subject is a subject requiring treatment or a subject with a disease or ailment. However, in other embodiments, a subject may be a healthy subject. The term does not indicate a specific age or sex. Therefore, it is intended to cover adult and newborn subjects, regardless of male or female.

[0125] The term "effective amount" refers to the amount of a compound that produces a therapeutic effect (e.g., sufficient to treat or prevent disease) in a patient receiving treatment. This effect can be objective (i.e., measurable by certain tests or biomarkers) or subjective (i.e., indicated or felt by the subject).

[0126] In further embodiments of the invention that may be mentioned, those in which the compounds of formula I are isotopically labeled are included. However, in other particular embodiments of the invention that may be mentioned, those in which the compounds of formula I are not isotopically labeled are included.

[0127] When used herein, the term "isotope labeling" includes referring to compounds of formula I, wherein a non-natural isotope (or a non-natural distribution of the isotope) is present at one or more sites in the compound. Those skilled in the art will understand that references herein to "one or more sites in the compound" refer to one or more atoms of a compound of formula I. Therefore, the term "isotope labeling" includes referring to compounds of formula I with isotope enrichment at one or more sites in the compound.

[0128] The isotopic labeling or enrichment of compounds of Formula I may be performed using radioactive or non-radioactive isotopes of any one of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine, and / or iodine. Specific isotopes that may be mentioned in this regard include... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 35 S, 18 F, 37 CI, 77 Br、 82 Br and 125 l).

[0129] When a compound of Formula I is labeled or enriched with a radioactive or non-radioactive isotope, the compound of Formula I that may be mentioned includes those in which at least one atom of the compound exhibits an isotopic distribution, wherein the radioactive or non-radioactive isotope of the atom in question is present at a level of at least 10% (e.g., 10% to 5000%, particularly 50% to 1000%, and more particularly 100% to 500%) exceeding the natural level of the radioactive or non-radioactive isotope.

[0130] Compounds of Formula I can be prepared for administration to subjects. Therefore, in another aspect of the invention, compositions are provided comprising compounds of Formula I as described herein, or pharmaceutically acceptable salts or solvates thereof, incorporated with one or more pharmaceutically acceptable adjuvants, diluents, and carriers.

[0131] Compounds of Formula I may be administered by any suitable route, but particularly by oral, intravenous, intramuscular, skin, subcutaneous, mucosal (e.g., sublingual or buccal), rectal, transdermal, nasal, pulmonary (e.g., tracheal or bronchial), local, or any other parenteral route, in the form of a pharmaceutically acceptable dosage form comprising the compound. Specific modes of administration that may be mentioned include oral, intravenous, skin, subcutaneous, nasal, intramuscular, or intraperitoneal administration.

[0132] Compounds of Formula I will generally be administered as a pharmaceutical formulation consisting of a mixture of a pharmaceutically acceptable adjuvant, diluent, or carrier, which may be chosen with due consideration of the intended route of administration and standard pharmaceutical practice. Such a pharmaceutically acceptable carrier may be chemically inert to the active compound and may not cause harmful side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations can be found, for example, in Remington, The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, parenteral-acceptable aqueous solutions may be used, which are free of pyrogens and have the necessary pH, isotonicity, and stability. Suitable solutions will be well-known to those skilled in the art, with numerous methods described in the literature. A brief overview of drug delivery methods can also be found, for example, in Langer, Science (1990) 249, 1527.

[0133] Otherwise, the preparation of a suitable formulation can be routinely carried out by technicians using conventional techniques and / or in accordance with standards and / or recognized pharmaceutical practices.

[0134] The amount of compound of formula I in any pharmaceutical preparation used according to the present invention will depend on various factors, such as the severity of the condition to be treated, the specific patient to be treated, and one or more compounds used. In any case, the amount of compound of formula I in the preparation can be routinely determined by a person skilled in the art.

[0135] For example, solid oral compositions such as tablets or capsules may contain 1 to 99% (w / w) of the active ingredient; 0 to 99% (w / w) of a diluent or filler; 0 to 20% (w / w) of a disintegrant; 0 to 5% (w / w) of a lubricant; 0 to 5% (w / w) of a flow aid; 0 to 50% (w / w) of a granulating agent or binder; 0 to 5% (w / w) of an antioxidant; and 0 to 5% (w / w) of a colorant. Controlled-release tablets may additionally contain 0 to 90% (w / w) of a release-controlled polymer.

[0136] Parenteral preparations (such as solutions or suspensions for injection or solutions for infusion) may contain 1 to 50% (w / w) of the active ingredient; and 50% (w / w) to 99% (w / w) of a liquid or semi-solid carrier or support (e.g., a solvent, such as water); and 0 to 20% (w / w) of one or more other excipients such as buffers, antioxidants, suspension stabilizers, tension modifiers, and preservatives.

[0137] Depending on the disease to be treated, the patient, and the route of administration, compounds of Formula I can be administered to patients in need at different therapeutically effective doses.

[0138] However, in the context of this invention, the dosage administered to mammals, particularly humans, should be sufficient to produce a therapeutic or diagnostic response in mammals within a reasonable timeframe. Those skilled in the art will recognize that the selection of the exact dosage and composition, as well as the most suitable delivery method, will also be particularly influenced by: the pharmacological properties of the formulation, the nature and severity of the condition being treated, the recipient's physical condition and mental sensitivity, the potency of the specific compound, the patient's age, condition, weight, sex, and response, and the stage / severity of the disease.

[0139] Administration can be continuous or intermittent (e.g., by bolus injection). Dosage can also be determined by timing and frequency of administration. In the case of oral or parenteral administration, the dose of the compound of formula I can vary from about 0.01 mg to about 1000 mg per day.

[0140] In any case, a licensed physician or other skilled worker will be able to routinely determine the actual dose that will be most appropriate for an individual patient. The doses mentioned above are examples of average cases; of course, there may be individual cases in which higher or lower dose ranges are to be expected, and such cases are within the scope of this invention.

[0141] The aspects and implementation of the invention will now be discussed with reference to the following non-limiting embodiments.

[0142] Example

[0143] Material

[0144] Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich. All amino acid derivatives were purchased from GL Biochem. PBr3 and p-aminobenzyl alcohol were purchased from Tokyo Chemical Industry Co., Ltd. Anti-mouse PD-L1 (B7-H1) (clone number: 10F.9G2, Cat No. BP0101) was purchased from Bio X Cell. Anti-mouse CD16 / 32 (Cat No. 101302), Alexa 700 anti-mouse CD45 (Cat No. 103128), FITC anti-mouse CD3 (Cat No. 100204), PE anti-mouse CD8a (Cat No. 100708), APC anti-human / mouse GrB recombinant antibody (Cat No. 372204), PerCP anti-mouse / human CD11b (Cat No. 101229), ultra-LEAF TM Purified anti-mouse CD47 antibody (Cat No. 127518), recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF, Cat No. 576304), recombinant rat interferon-γ (IFN-γ, Cat No. 598802), recombinant rat interleukin-4 (IL-4, Cat No. 776902), intracellular staining permeation wash buffer (ISPWB), ACK lysis buffer, PE anti-mouse / human CD11b, and APC anti-mouse Ly-6G were purchased from Biolegend. Neutrophil elastase (NE) polyclonal antibody (PA5-79198), anti-Mo Ly-6G APC (17-9668-82), anti-mouse NOS2 PE (12-5920-82), live / dead... TM (Live / Dead TM Fixable blue dead cell strains (Cat No. L23105), Dynabeads TM FlowComp TM Mouse CD8 assay kit (Cat No. 11462D) and second antibody Alexa 488-conjugated goat anti-rabbit IgG (Cat No. 2045215) was purchased from Thermo Fisher Scientific. Caspase-1 (D-3) Alexa 647 and F4 / 80 (C-7) Alexa 488 (sc-377009) was purchased from Santa Cruz Biotechnology. GrB was purchased from Novoprotein. Caspase-1 was purchased from BioVision. Cathepsin C (DPPI) and APN were obtained from R&D systems. NE, LPS, bovine serum albumin (BSA), HEPES, CHAPS (3-[(3-cholamidopropyl)dimethylamino]propanesulfonic acid inner salt), PBS, heat-inactivated horse serum, interleukin-2 (IL-2), and deoxyribonuclease I were purchased from Sigma-Aldrich. MTS solution was purchased from PROMEGAPTE LTD. Duchenne modified Eagle medium (DMEM), fetal bovine serum (FBS), Iscofer modified Duchenne medium (IMDM), and Roswell Park Memorial Institute medium (RPMI) were purchased from GIBCO. OCT medium was purchased from Sakura Fineteck Japan. Heparinized capillaries were purchased from Greiner Bio-One GmbH. Type I and Type IV collagenase were purchased from Thermofisher. Dialysis bags were used for dialysis. Dialysis bags were purchased from USAViskase.

[0145] Mouse breast cancer cell line 4T1, mouse colorectal cancer cell line CT26, mouse embryonic fibroblast 3T3, mouse neutrophil MPRO, and mouse macrophage cell line RAW 264.7 were purchased from the American Type Culture Collection (ATCC).

[0146] Analytical techniques

[0147] UV-Vis spectroscopy

[0148] UV-Vis spectra were recorded on a Shimadzu UV-2450 spectrophotometer.

[0149] Fluorescence spectroscopy

[0150] Fluorescence spectra were recorded on a Fluorolog 3-TCSPC fluorescence spectrophotometer (Horiba Jobin Yvon).

[0151] HPLC

[0152] HPLC profiles were measured on an Agilent 1260 system equipped with a UV detector, a G1311B pump, and an Agilent Zorbax SB-C18RP (9.4 × 250 mm) column, with CH3OH containing trifluoroacetic acid (TFA, 0.1%) and water containing TFA (0.1%) as eluents.

[0153] Table 1. Enzyme kinetic parameters of TASMR and CyA.

[0154]

[0155] Table 2. HPLC conditions for enzymatic analysis.

[0156]

[0157]

[0158] Proton nuclear magnetic resonance (NMR) 1 ¹H NMR spectroscopy

[0159] Recorded on Bruker 400MHz NMR 1 H NMR spectrum.

[0160] Liquid Chromatography-Mass Spectrometry (LCMS)

[0161] LCMS spectra were measured on a Thermo Finnigan Polaris Q quadrupole ion trap mass spectrometer equipped with a standard electrospray ionization (ESI) source.

[0162] Fluorescence imaging

[0163] Fluorescence imaging of cells was obtained using a laser scanning microscope LSM800 (Zeiss).

[0164] In vivo animal fluorescence imaging

[0165] In vivo fluorescence images of animals were captured using an IVIS imaging system (IVIS-CT machine, PerkinElmer), and the target region was analyzed using Living Image 4.3 software.

[0166] tissue section

[0167] Tissue sections were obtained using a cryostat (Leica).

[0168] General procedures for purification by high performance liquid chromatography (HPLC)

[0169] HPLC purification was performed on an Agilent 1260 gradient preparation system equipped with a G1361A pump, a UV detector and an Agilent Zorbax SB-C18RP (21.2 × 150 mm) column, with CH3OH containing 0.1% TFA and water containing 0.1% TFA as eluents (Tables 2 and 3).

[0170] Table 3. HPLC conditions used for purifying TASMR and their precursors.

[0171]

[0172]

[0173] General procedures for cell culture

[0174] All cells were cultured at 37°C in a humid environment containing 5% CO2 and 95% air. 3T3 and RAW264.7 cells were cultured in DMEM containing 10% FBS. 4T1 and CT26 cells were cultured in RPMI 1640 containing 10% FBS. MPRO cells were cultured in IMDM containing 4 mM L-glutamine, with the adjusted IMDM containing 1.5 g / L sodium bicarbonate (containing 10 ng / mL mouse GM-CSF) and 20% heat-inactivated horse serum. [The last sentence appears to be incomplete and requires further context.] Untouched TM CD8 T cells were isolated from the spleen of BALB / c mice using a mouse CD8 cell kit and cultured in RPMI 1640 containing IL-2. Bone marrow-derived dendritic cells (BMDCs) were isolated from the bone marrow of BALB / c mice according to a previous protocol (Madaan, A. et al., J. Biol. Methods 2014, 1, e1) and cultured in RPMI 1640 containing GM-CSF (20 ng / mL). RAW264.7 cells were polarized into M1 macrophages by incubation with LPS (10 pg / mL) and IFN-γ (20 ng / mL) for 24 h. RAW264.7 cells were polarized into M2 macrophages by incubation with IL-4 (20 ng / mL) for 24 h. Both M1 and M2 macrophages were cultured in DMEM.

[0175] Establish a universal procedure for tumor-bearing mice.

[0176] All mouse experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanyang Technological University (NTU). Six-week-old female BALB / c mice were purchased from InVivos in Singapore. To establish immunogenic tumors, 4T1 cancer cells in PBS were injected at a concentration of 1 × 10⁻⁶. 6 CT26 cancer cells were subcutaneously implanted into the right flank of mice at a density of 10 cells / mouse. To establish a highly immunogenic tumor, CT26 cancer cells in PBS were injected at a density of 2 × 10⁻⁶ cells / mouse. 6 A density of 1 cell / mouse was subcutaneously implanted into the right side of the mouse's back.

[0177] Statistical and reproducibility

[0178] The in vivo and in vitro fluorescence intensities of the target region were quantified using Living Image 4.3 software. Statistical comparisons between the two groups were determined by a two-tailed Student's t-test. For statistical analysis, P < 0.05 was considered statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered statistically significant.

[0179] Example 1. Synthesis of TAMR

[0180] The TAMR comprises three key units: a tumor passive targeting component, a fluorescent signal transduction component, and a dual-locked TIL response component, which can only be fully cleaved in the presence of both cancer and leukocytes (Figure 1). The TAMR is inherently non-fluorescent and only activates their NIRF signaling in the presence of both cancer and leukocyte biomarkers. This dual-locked tandem design ensures that signal activation is triggered solely by the TIL, and not by leukocytes in other normal and inflammatory tissues. Synthetic TAMR M1 TAMR CTL and TAMR NE Real-time dynamic imaging of TIME was achieved by detecting M1 macrophages, CTLs, and neutrophils separately. To eliminate differences in TAMR accumulation in tumors, PVP-IR800 was synthesized as an "always-on" reference probe and used for normalization during signal analysis.

[0181] Synthesis of PVP-IR800

[0182] IR800-N3 (5 mg, 0.004 mmol) and PVP-acetylene (12 mg, 0.004 mmol) were dissolved in DMSO (1.5 mL). Then, CuSO4·5H2O (1 mg, 0.004 mmol) and sodium ascorbate (1.6 mg, 0.008 mmol) were added to deionized water (1.5 mL). The reaction mixture was stirred at 25 °C for 12 h. The reaction solution was then dialyzed against deionized water for 24 h to remove salts and DMSO, and lyophilized to obtain PVP-IR800.

[0183] TAMR was constructed on a near-infrared (NIR) hemicyanine dye (CyOH) using the following three steps (Figure 2). First, a peptide substrate cleaved by the cancer biomarker (APN) alanine (A) was conjugated with CyOH to generate CyA. Then, a leukocyte-biomarker-reactive peptide substrate, comprising the following components, was conjugated with CyA to provide the TAMR precursor TASMR. M1 TASMR CTL and TASMR NE Cas-1 cleavable peptide substrates for M1 macrophages: N-acetyl-Tyr-Val-Ala-Asp-OH (YVAD); GrB cleavable peptide substrates for CTLs: N-acetyl-Ile-Glu-Phe-Asp-OH (IEFD); and NE cleavable peptide substrates for neutrophils: N-methoxysuccinyl-Ala-Ala-Pro-Val (AAPV). For TASMR... M1 The NO-cleavable substrate OPD is conjugated with the carboxyl (COOH) side chain group of YVAD to enhance its specificity for M1 macrophages. Finally, the TAMR precursor is clicked with an alkyne-functionalized PVP to obtain TAMR. Therefore, the tandem design is modular and can be generalized to different TILs, such as TAMR detection of tumor-infiltrating M1 macrophages, CTLs, and neutrophils respectively. M1 TAMR CTL and TAMR NE The composite is shown in Figure 2.

[0184] Synthesis of CyOH

[0185] CyCl was synthesized according to a previous protocol (Huang, J. et al., Nat. Mater. 2019, 18, 1133-1143). K₂CO₃ (1382 mg, 10 mmol) and resorcinol (1101 mg, 10 mmol) were dissolved in CH₃CN (10 mL), and the mixture was stirred at 55 °C for 20 min. Then, a CH₃CN solution of CyCl (16.25 mg, 5 mmol) was added to the reaction mixture. The reaction mixture was stirred at 55 °C for another 6 h, after which CH₃CN was removed. The crude product was purified by silica gel column chromatography using DCM and methanol (CH₃OH) (DCM / CH₃OH = 30 / 1) to obtain CyOH in 79% yield.

[0186] MS of CyOH: m / z 467.36. 1 H NMR (400MHz, MeOD): δ (ppm): 8.38 (d, J = 12.0 Hz, 1H), 7.59 (s, 1H), 7.49 (d, J = 8, 1H), 7.38 (t, 2H), 7.21 (t, 2H), 6.73 (d, J = 8, 1H), 6.5 7(s,1H),6.05(d,J=16,1H),4.07(t,2H),3.45(t,2H),2.74(t,2H),2.68(t,2H),1.91-1.89(m,4H),1.79-1.74(m,2H),1.74(s,6H).

[0187] PVP Synthesis

[0188] N-vinylpyrrolidone (1 g, 9 mmol) and AIBN (20 mg) were dissolved in isopropoxyethanol (10 mL), followed by rinsing with nitrogen for 10 min. The reaction mixture was stirred at 60 °C for 4 h under nitrogen protection. After completion, the isopropoxyethanol was concentrated, and the residue was precipitated in excess diethyl ether (200 mL). The white precipitate was centrifuged at 7500 rpm for 10 min and dried under vacuum at 37 °C to obtain PVP in 67% yield.

[0189] 1 H NMR (400MHz, MeOD): δ (ppm): 3.92-3.74 (m, 32H), 3.29 (m, 54H), 2.38-2.27 (m, 54H), 2.06-2.01 (m, 54H), 1.75-1.46 (m, 60H).

[0190] Synthesis of PVP-Alkynes

[0191] PVP (3 g, 1 mmol) was dissolved in anhydrous THF (40 mL), and NaH (240 mg, 10 mmol) was added. After 10 min of bubble generation, 3-bromopropyne (1.19 g, 10 mmol) was rapidly added to the reaction solution, followed by stirring at 25 °C for 24 h. After completion, the THF was concentrated, and the mixture in the THF was precipitated in excess diethyl ether (200 mL). The white precipitate was centrifuged at 7500 rpm for 10 min. The residue was further dissolved in water and dialyzed against deionized water for 4 h to remove salt (MWCO = 1000), and then freeze-dried to obtain PVP-propyne in 77% yield.

[0192] 1 H NMR (400MHz, MeOD): δ (ppm): 4.17 (s, 2H), 3.92-3.74 (m, 32H), 3.29 (m, 54H) ,2.51(s,2H),2.38-2.27(m,54H),2.06-2.01(m,54H),1.75-1.46(m,60H).

[0193] Synthesis of Ac-Y(tBu)VAD(OtBu)-OH, Ac-IE(OtBu)FD(OtBu)-OH and MeOSuc-AAPV-OH

[0194] The peptides Ac-Y(tBu)VAD(OtBu)-OH, Ac-IE(OtBu)FD(OtBu)-OH and MeOSuc-AAPV-OH were prepared by solid-phase peptide synthesis (SPPS, OAMusaimi, BGde la Torre & F. Albericio, GreenChem. 2020, 22, 996-1018).

[0195] Ac-Y(tBu)VAD(OtBu)-OH

[0196]

[0197] 1 H NMR (400MHz, MeOD): δ (ppm): 7.18 (d, J = 8, 2H), 6.92 (d, J = 8, 2H), 4.75 (t, 1H), 4.67-4.63 (m, 1H), 4.44-4.39 (m, 1H), 4.23-4.19 (m, 1H),3.14-3.09(m,1H),2.88-2.82(m,2H),2.77(d,J=4,2H),1.92(s,3H),1.46(s,9H),1.40(d,J=8,3H),1.33(s,9H),0.98(t,6H).

[0198] Ac-IE(OtBu)FD(OtBu)-OH

[0199]

[0200] 1 H NMR (400MHz, MeOD): δ (ppm): 7.26-7.15 (m, 5H), 4.76 (t, 1H), 4.67-4.63 (m, 1H), 4.32-4.28 (m, 1H), 4.15 (d, J = 8, 1H), 3.25 (m, 2H), 3.2 (m, 1H), 2.98-2.92(m,1H),2.79-2.70(m,2H),2.30-2.09(m,2H),2.01(s,3H), 1.97-1.90(m,1H),1.86-1.75(m,2H),1.45(s,18H),0.93-0.83(m,6H).

[0201] MeOSuc-AAPV-OH

[0202]

[0203] 1 H NMR (400MHz, MeOD): δ (ppm): 4.64-4.52 (m, 2H), 4.35 (t, 1H), 4.29 (t, 1H), 3.81-3.75 (m, 1H), 3.66 (s, 3H), 3.32-3.30 (m ,1H),2.69-2.58(m,2H),2.55-2.45(m,2H),2.21-2.18(m,2H),2.04-1.91(m,3H),1.36-1.32(m,6H),0.99(d,J=8,6H).

[0204] Synthesis of Fmoc-A-PABA

[0205] EEDQ (742 mg, 3.0 mmol), PABA (369 mg, 3.0 mmol), and Fmoc-A-OH (311.3 mg, 1.0 mmol) were dissolved in DCM (15 mL), and the reaction mixture was continuously stirred at 25 °C for 6 h. The residue was purified by HPLC and lyophilized to obtain Fmoc-A-PABA in 91% yield.

[0206] 1H NMR (400MHz, MeOD): δ (ppm): 7.82 (d, J = 8, 2H), 7.70 (t, 2H), 7.56 (d, J = 8, 2H), 7.40 (t, 2H) ), 7.33 (d, J = 8, 4H), 4.58 (s, 2H), 4.41 (d, J = 8, 2H), 4.29-4.22 (m, 2H), 1.44 (d, J = 8, 3H).

[0207] Synthesis of Cy-A-Fmoc

[0208] Fmoc-A-PABA (100 mg, 0.24 mmol) was dissolved in anhydrous THF, followed by the addition of PBr3 (200 mg, 0.72 mmol). The reaction mixture was stirred at 0 °C for 2 h. Then, THF was removed, and the residue was dissolved in excess ethyl acetate (EA, 200 mL). The solution was washed three times with an aqueous solution of NaHCO3. After concentration and drying, the residue was dissolved in anhydrous CH3CN (50 mL), followed by the addition of CyOH (37.4 mg, 0.08 mmol) and N,N-diisopropylethylamine (DIPEA, 40 μL). The reaction mixture was stirred at 55 °C for 8 h. Then, CH3CN was removed, and the residue was purified by HPLC and lyophilized to give Cy-A-Fmoc in 97% yield.

[0209] 1 H NMR (400MHz, CDCl3): δ (ppm): 8.66 (d, J = 12, 1H), 7.75 ( d, J = 8, 2H), 7.67 ( d, J = 8, 3H) ,7.60(t,2H),7.46-7.40(m,6H),7.32(t,3H),7.24(t,2H),7.01-6.98(m,2H),6.44 (d,J=16,1H),5.22(s,2H),4.31-4.23(m,5H),4.10(t,1H),3.40(t,2H),2.71(t,2H ), 2.65 (t, 2H), 1.89-1.86 (m, 4H), 1.75 (s, 6H), 1.74-1.7 (m, 2H), 1.41 (d, J = 8, 3H).

[0210] Synthesis of CyA

[0211] Cy-A-Fmoc (10 mg, 0.012 mmol) was dissolved in DMF (2 mL). Piperidine (100 μL) was added to the solution, followed by stirring at 25 °C for 5 min. The mixture was purified by HPLC and lyophilized to obtain CyA in 88% yield.

[0212] CyA MS: m / z 643.43. 1 H NMR (400MHz, CDCl3): δ (ppm): 8.82 (d, J = 16, 1H), 7.91 ( d, J = 8, 2H), 7.75 ( d, J = 8, 2H), 7.7 1-7.68(m,2H),7.57(d,J=4,1H),7.53-7.48(m,3H),7.15(d,J=4,1H),7.09-7.05(m,1H), 6.56(d,J=12,1H),5.27(s,2H),4.51(t,1H),4.40(t,1H),4.12-4.07(m,1H),3.17(t,2H) ,3.0(t,2H),2.81(t,1H),2.75(t,1H),1.86(s,6H),1.83-1.77(m,6H),1.63(d,J=8,3H).

[0213] Synthesis of CyA-D(OtBu)AVY(tBu)

[0214] CyA (10 mg, 0.016 mmol), Ac-Y(tBu)VAD(OtBu)-OH (29.8 mg, 0.036 mmol), HBTU (12.1 mg, 0.032 mmol), HOBt (4.3 mmol, 0.032 mmol), and DIPEA (4.1 mg, 0.032 mmol) were dissolved in DMF (5 mL), and the mixture was stirred at 25 °C for 2 h. The reaction mixture was then purified by HPLC and lyophilized to obtain CyA-D(OtBu)AVY(tBu) in 91% yield.

[0215] 1H NMR (400MHz, CDCl3): δ (ppm): 8.79 (d, J = 16, 1H), 8.21 ( t, 1H), 8.15 ( d, J = 8, 1H), 8.00 ( d, J = 12, 3H), 7.74-7.72 (m, 2H), 7.63-7.57 (m, 2H) ),7.50-7.43(m,3H),7.17(d,J=8,1H),7.06-6.98(m,3H),6.55(d,J=16,1H),5.27(s,2H),4.67-4.60(m,1H),4.47-4.37(m,2H),4.31-4 .24(m,2H),4.11-4.08(m,2H),3.46(t,2H),3.19(s,2H),3.09-3.05(m,1H),2.81-2.70(m,6H),2.21(t,1H),2.11-2.03(m,2H),2.01-1. 97(m,2H),1.93(s,3H),1.83(s,6H),1.80-1.77(m,1H),1.48(d,J=8,3H),1.39(s,9H),1.35(d,J=8,3H),1.31(s,9H),1.02-0.9(m,6H).

[0216] Synthesis of CyA-DAVY

[0217]

[0218] CyA-D(OtBu)AVY(tBu) (8 mg) was dissolved in TFA (0.95 mL), followed by the addition of H2O (0.05 mL). The reaction was stirred at 0 °C and monitored by HPLC. After confirming complete deprotection of the tBu and OtBu groups, a saturated aqueous solution of NaHCO3 was added dropwise to neutralize the TFA. The mixture was then extracted with DCM. After drying with anhydrous Na2SO4, the DCM was removed, and the residue was purified by HPLC and freeze-dried to obtain the product CyA-DAVY in 52% yield.

[0219] MS of CyA-DAVY: m / z 1133.56. 1H NMR (400MHz, CDCl3): δ (ppm): 8.78 (d, J = 12, 1H), 8.21 (t, 1H), 7.95-7.91 ( m,1H),7.76-7.71(m,3H),7.58-7.55(m,2H),7.50-7.43(m,4H),7.39-7.3 3(m,2H),7.05(t,2H),6.70(d,J=8,1H),6.54(d,J=12,1H),5.27(s,2H),4 .65-4.62(m,1H),4.59-4.55(m,1H),4.47-4.44(m,1H),4.39(t,2H),4.26( t,1H),4.10(t,1H),3.26-3.15(m,2H),3.04-2.99(m,1H),2.94-2.92(m,1 H),2.91-2.89(m,1H),2.87(t,2H),2.83-2.77(m,2H),2.76-2.72(m,2H), 2.23-2.18(m,1H),2.08-2.05(m,2H),2.0-1.97(m,2H),1.93(s,3H),1.83 (s,6H),1.71(m,1H),1.48(d,J=8,3H),1.35(d,J=8,3H),1.02-0.9(m,6H).

[0220] TASMR M1 Synthesis

[0221] CyA-DAVY (10 mg, 0.009 mmol), OPD (4.9 mg, 0.09 mmol), HBTU (6.8 mg, 0.018 mmol), HOBt (2.4 mg, 0.018 mmol), and DIPEA (2.3 mg, 0.018 mmol) were dissolved in DMF (3 mL). The reaction mixture was stirred at 25 °C for 0.5 h. The reaction mixture was then purified by HPLC and lyophilized to obtain TASMR. M1 The yield was 41%.

[0222] TASMR M1 MS: m / z 612.89. 1H NMR (400MHz, CDCl3): δ (ppm): 8.76 (d, J = 12, 1H), 8.00 (m, 3H), 7.78-7.72 (m, 3H), 7.64-7.56 (m, 3H), 7.50-7.42 (m, 5H), 7. 09-6.98(m,4H),6.70(d,J=16,2H),6.55(d,J=16,1H),5.28(s,2H),4.59-4.55(m,1H),4.41-4.35(m,3H),4.33-4.24(m,2 H),4.11(t,1H),3.46(t,2H),3.15-3.08(m,2H),2.95-2.91(m,1H),2.83-2.78(m,2H),2.76-2.72(m,3H),2.65-2.59(m,1 H),2.23-2.18(m,2H),2.08-2.03(m,4H),1.93(s,3H),1.83(s,6H),1.46(d,J=8,3H),1.37(d,J=8,3H),1.02-0.9(m,6H).

[0223] Synthesis of CyA-D(OtBu)FE(OtBu)I

[0224] CyA (10 mg, 0.016 mmol), Ac-IE(OtBu)FD(OtBu)-OH (21.7 mg, 0.032 mmol), HBTU (12.1 mg, 0.032 mmol), HOBt (4.3 mmol, 0.032 mmol), and DIPEA (4.1 mg, 0.032 mmol) were dissolved in DMF (5 mL). The reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was then purified by HPLC and lyophilized to give CyA-D(OtBu)FE(OtBu)I in 91% yield.

[0225] 1H NMR (400MHz, CDCl3): δ (ppm): 8.78 (d, J = 16, 1H), 8.36-8.0 (m, 2H), 7.75 (t, 2H), 7.57-7.55 (m, 2H), 7.47 (t, 3H), 7.42 (s, 1 H),7.28-7.22(m,5H),7.08-7.05(m,2H),6.54(d,J=12,1H),5.28(s,2H),4.65(t,1H),4.56-4.53(m,1H),4.43-4.37(m,3H ),4.23-4.20(m,1H),4.14-4.11(m,1H),3.47(t,2H),3.25-3.20(m,2H),3.06-3.0(m,1H),2.83-2.72(m,7H),2.25-2.14( m,2H),2.07(s,3H),1.97-1.93(m,5H),1.84-1.76(m,8H),1.48(s,3H),1.41(s,18H),1.3-1.19(m,2H),0.96-0.94(m,6H).

[0226] TASMR CTL Synthesis

[0227] CyA-D(OtBu)FE(OtBu)I (6.5 mg) was dissolved in TFA (0.95 mL), followed by the addition of H2O (0.05 mL). The reaction was stirred at 0 °C and monitored by HPLC. After confirming complete deprotection of the OtBu group, a saturated aqueous solution of NaHCO3 was added dropwise until trifluoroacetic acid was neutralized. The mixture was then extracted with DCM. After drying with anhydrous Na2SO4, the DCM was removed, and the residue was purified by HPLC and freeze-dried to obtain the product TASMR. CTL The yield was 58%.

[0228] TASMR CTL MS: m / z 1189.6. 1H NMR (400MHz, CDCl3): δ (ppm): 8.77 (d, J = 16, 1H), 7.98 (s, 1H), 7.73-7.62 (m, 3H), 7.54-7.51 (m, 2H), 7.47-7.25 (m, 4H), 7.24-7.19 (m, 5H),7.04-6.96(m,2H),6.52(d,J=12,1H),5.26(s,2H),4.63(t,1H),4.54-4.51(m,1H),4.42-4.34(m,2H),4.31-4.22(m,2H),4.11(d, J=8,1H),3.46(t,2H),3.21-3.13(m,2H),3.03-2.97(m,3H),2.86-2.8(m,3H),2.78-2.74(m,1H),2.73-2.71(m,1H),2.35-2.28(m,2H ),2.19(t,1H),2.04(s,3H),1.97-1.93(m,3H),1.84-1.76(s,6H),1.46-1.4(m,3H),1.48(s,3H),1.3-1.19(m,2H),0.96-0.94(m,6H).

[0229] TASMR NE Synthesis

[0230] CyA (10 mg, 0.016 mmol), MeOSuc-AAPV-OH (15.0 mg, 0.032 mmol), HBTU (12.1 mg, 0.032 mmol), HOBt (4.3 mg, 0.032 mmol), and DIPEA (4.1 mg, 0.032 mmol) were dissolved in DMF (5 mL). The reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was then purified by HPLC and lyophilized to obtain TASMR. NE The yield was 91%.

[0231] 1H NMR (400MHz, CDCl3): δ (ppm): 8.78 (d, J = 12, 1H), 7.71-7.67 (m, 4H), 7.51-7.42 (m, 6H) ),7.1(t,2H),6.53(d,J=12,1H),5.25(s,2H),4.55-4.26(m,7H),3.71-3.57(m,2H),3 .5-3.45(m,2H),3.41(t,3H),2.99-2.72(m,4H),2.6-2.5(m,4H),2.22-2.1(m,3H),2 .0-1.95(m,5H),1.82(s,6H),1.45-1.4(m,2H),1.35-1.27(m,9H),0.96-0.94(m,6H).

[0232] TAMR M1 TAMR CTL and TAMR NE Synthesis

[0233] TASMR M1 (12 mg, 0.01 mmol) or TASMR CTL (12 mg, 0.01 mmol) or TASMR NE 11 mg (0.01 mmol) and PVP-acetylene (30 mg, 0.01 mmol) were dissolved in DMSO (1.5 mL). Then, CuSO4·5H2O (2.5 mg, 0.01 mmol) and sodium ascorbate (4 mg, 0.02 mmol) were added to deionized water (1.5 mL). The reaction mixture was stirred at 25 °C for 12 h. The reaction solution was then dialyzed against deionized water for 24 h to remove salts and DMSO, and then freeze-dried to obtain TAMR. M1 TAMR CTL and TAMR NE Their yields were 77%, 92%, and 88%, respectively.

[0234] TAMR M1 . 1H NMR(400MHz,CDCl3):δ(ppm):8.76(d,J=12,1H),8.00(m,3H),7.82(s,1H),7.78-7.72(m,3H),7.64-7.56(m,3H),7.50-7.42(m,5H),7.09-6.98(m,4H),6.70(d,J=16,2H),6.55(d,J=16,1H),5.28(s,2H),4.59-4.55(m,1H),4.41-4.35(m,3H),4.33-4.24(m,2H),4.15(s,2H),4.11(t,1H),3.92-3.74(m,32H),3.46(t,2H),3.29(m,54H),3.15-3.08(m,2H),2.95-2.91(m,1H),2.83-2.78(m,2H),2.76-2.72(m,3H),2.65-2.59(m,1H),2.38-2.27(m,54H),2.23-2.18(m,2H),2.08-2.03(m,58H),1.93(s,3H),1.83(s,6H),1.75-1.46(m,60H),1.45(d,J=8,3H),1.37(d,J=8,3H),1.02-0.9(m,6H)。

[0235] TAMR CTL 。 1H NMR(400MHz,CDCl3):δ(ppm):8.77(d,J=16,1H),7.98(s,1H),7.82(s,1H),7.73-7.62(m,3H),7.54-7.51(m,2H),7.47-7.25(m,4H),7.24-7.19(m,5H),7.04-6.96(m,2H),6.52(d,J=12,1H),5.26(s,2H),4.63(t,1H),4.54-4.51(m,1H),4.42-4.34(m,2H),4.31-4.22(m,2H),4.15(s,2H),4.11(d,J=8,1H),3.92-3.74(m,32H),3.46(t,2H),3.29(m,54H),3.21-3.13(m,2H),3.03-2.97(m,3H),2.86-2.8(m,3H),2.78-2.74(m,1H),2.73-2.71(m,1H),2.35-2.28(m,56H),2.19(t,1H),2.06-2.01(m,57H),1.97-1.93(m,3H),1.84-1.76(s,6H),1.75-1.46(m,60H),1.46-1.4(m,3H),1.48(s,3H),1.3-1.19(m,2H),0.96-0.94(m,6H)。

[0236] TAMR NE 。 1 H NMR(400MHz,CDCl3):δ(ppm):8.78(d,J=12,1H),7.82(s,1H),7.71-7.67(m,4H),7.51-7.42(m,6H),7.1(t,2H),6.53(d,J=12,1H),5.25(s,2H),4.55-4.26(m,7H),4.15(s,2H),3.92-3.74(m,32H),3.71-3.57(m,2H),3.5-3.45(m,2H),3.41(t,3H),3.29(m,54H),2.99-2.72(m,4H),2.6-2.5(m,4H),2.38-2.27(m,54H),2.22-2.1(m,3H),2.06-2.01(m,54H),2.0-1.95(m,5H),1.82(s,6H),1.75-1.46(m,60H),1.45-1.4(m,2H),1.35-1.27(m,9H),0.96-0.94(m,6H)。

[0237] Synthesis of PEG-Cy

[0238] CyOHN3 (6 mg, 0.01 mmol) and PEG-acetylene (20 mg, 0.01 mmol) were dissolved in DMSO (1.5 mL). Then, CuSO4·5H2O (2.5 mg, 0.01 mmol) and sodium ascorbate (4 mg, 0.02 mmol) were added to deionized water (1.5 mL). The reaction mixture was stirred at 25 °C for 12 h. The reaction solution was then dialyzed against deionized water for 24 h to remove salts and DMSO, and then freeze-dried to obtain PEG-Cy.

[0239] Synthesis of PVP-Cy

[0240] CyOHN3 (6 mg, 0.01 mmol) and PVP-acetylene (30 mg, 0.01 mmol) were dissolved in DMSO (1.5 mL). Then, CuSO4·5H2O (2.5 mg, 0.01 mmol) and sodium ascorbate (4 mg, 0.02 mmol) were added to deionized water (1.5 mL). The reaction mixture was stirred at 25 °C for 12 h. The reaction solution was then dialyzed against deionized water for 24 h to remove salts and DMSO, and then freeze-dried to obtain PVP-Cy.

[0241] Pharmacokinetic studies

[0242] Mice were intravenously injected with unlockable PEG-Cy (5 μmol / kg) and PVP-Cy (5 μmol / kg). Blood samples from mice injected with PEG-Cy were collected using heparinized capillaries at 1, 4, 7, 11, 16, 25, 35, 55, 75, 95, 120, and 150 min after PEG-Cy injection. Blood samples from mice injected with PVP-Cy were collected using heparinized capillaries at 1, 4, 7, 11, 16, 25, 35, 55, 75, 95, 120, 150, and 180 min after PVP-Cy injection. The blood samples in the heparinized capillaries were then centrifuged at 3500 rpm for 10 min and subsequently quantified by HPLC.

[0243] Results and discussion

[0244] PVP was chosen as the passive tumor targeting component because we demonstrated that it has a higher tumor accumulation efficiency (1.5 times) and a longer circulating half-life (4.4 times) compared to PEG. Figure 3 ).

[0245] Example 2. Characterization of TAMR

[0246] The TAMR and TASMR prepared in Example 1 were characterized.

[0247] Optical Measurement

[0248] TAMR (25 μM) was incubated with its corresponding leukocyte biomarker or a combination of tumor and corresponding leukocyte biomarker (50 μM diethylamine nonanoate, 1 U Cas-1, 0.5 μg APN, 0.5 μg GrB, 2.5 mUNE) in the appropriate buffer at 37 °C for 2 h; for TAMR M1 Enzymatic experiments were performed in Cas-1 HEPES buffer (50 mM HEPES, pH 7.2, 50 mM NaCl, 0.1% CHAPS, 5% glycerol, 10 mM EDTA, 10 mM dithiothreitol (DTT)). For TAMR... CTL Enzymatic experiments were performed in Tris buffer (100 mM Tris, pH 7.5, 150 mM NaCl). GrB (0.5 μg) was first activated with cathepsin C (0.2 μg) in MES buffer (50 mM MES, pH 5.5, 50 mM NaCl) for 4 h. For TAMR NE The enzymatic experiments were performed in NE Tris buffer (50 mM Tris, 1 M NaCl, 0.05% (w / v) Brij-35, pH 7.5). After completion, the UV-Vis absorption and fluorescence spectra of the enzymatically catalyzed solution were recorded.

[0249] In vitro selective studies

[0250] TAMR or TASMR (25 μM) was incubated with various enzymes, including uPA (0.5 μg), NTR (0.5 μg), GGT (0.5 μg), FAP (0.2 mU), caspase-3 (0.5 μg), CTSS (0.5 μg), GrB (0.5 μg), NE (0.5 mU), APN (0.5 μg), Cas-1 (1 U), and cathepsin C (0.5 μg), as well as combinations of some of these enzymes, in their respective buffers at 37 °C for 2 h. For NTR, GGT, and caspase-3, enzymatic assays were performed in PBS (10 mM, pH 7.4) buffer. For FAP, enzymatic assays were performed in HEPES buffer (50 mM HEPES, pH 7.4, 0.1% bovine serum albumin (BSA), 5% glycerol). For cathepsin S, the enzymatic assay was performed in NaOAc buffer (NaOAc 50 mM, pH 5.5, 5 mM DTT, 250 mM NaCl). For APN, the enzymatic assay was performed in Tris buffer (50 mM Tris, pH 7.0). After completion, the fluorescence intensity of the enzymatically catalyzed solutions was measured using an IVIS spectral imaging system. Excitation: 675 nm. Emission: 720 nm.

[0251] Enzyme kinetics assay

[0252] TASMR at different concentrations M1 TASMR (2, 4, 8, 20, 40, 80, 160, or 200 μM) was incubated with Cas-1 (0.5 U) in HEPES buffer (50 mM HEPES, pH 7.2, 50 mM NaCl, 0.1% CHAPS, 10 mM EDTA, 5% glycerol, 10 mM DTT) at 37°C for 1 h. Different concentrations of TASMR were then incubated. CTL (5, 10, 20, 40, 80, or 120 μM) and GrB (0.25 μg) were incubated in Tris buffer (100 mM Tris, pH 7.5, 150 mM NaCl) at 37 °C for 30 min. Different concentrations of TASMR were then incubated. NECyA (5, 10, 20, 40, 80, 120, 160, or 200 μM) and NE (2.5 mU) were incubated with Tris buffer (50 mM Tris, 1 M NaCl, 0.05% (w / v) Brij-35, pH 7.5) at 37 °C for 4 min. CyA (10, 20, 40, 80, 120, or 200 μM) and APN (0.25 μg) were incubated with Tris buffer (50 mM Tris, pH 7.0) at 37 °C for 20 min. After incubation, the mixture was measured by HPLC. Calculate the enzyme-catalyzed reaction rate (nmol / min or pmol / s), plot it as a function of TASMR or CyA concentration, and fit it to the Michaelis-Menten equation: V = Vmax * [S] / (Km + [S]), where Vmax represents the maximum theoretical reaction rate, [S] represents the substrate concentration, and Km represents the Michaelis constant.

[0253] Results and discussion

[0254] TAMR ( M1、CTL、NE They exhibit similar optical properties, with absorption peaks located at ~610 and 660 nm, respectively. Figure 4 The TAMR absorbance at 610 nm decreased only in the presence of both the tumor and the corresponding leukocyte biomarker, with the appearance of a new peak at ~680 nm belonging to the unlocked CyOHP; furthermore, fluorescence at ~710 nm increased 12 to 18 times (Fig. 5a-c). In contrast, no change in absorption or fluorescence was observed after incubation with either single biomarker. Similar optical curves were also observed for TASMR. Figure 6 More importantly, the fluorescence intensity of TAMR and TASMR showed negligible enhancement after incubation with other interfering enzymes (Figs. 5d-f and 7), indicating their high specificity.

[0255] HPLC analysis was used to investigate the structural changes of TASMR (TAMR precursors) in response to their respective biomarkers. TASMR was used instead of TAMR because the HPLC traces of TAMR remained similar before and after enzymatic activation due to the presence of PVP, which dominates the elution properties. Incubation of TASMR with both cancer and leukocyte biomarkers resulted in elution peaks attributable to CyOH (T0). R =17.8 min), which was undetectable after incubation with any single biomarker. Figure 8 The catalytic efficiencies (Kcat / Km) of Cas-1, GrB, and NE for their respective TASMR values ​​were determined to be 0.003, 0.002, and 0.49 μM, respectively.-1 s -1 ( Figure 9 (ac and Table 1). The TASMR product CyA, after being cleaved by the corresponding leukocyte biomarker, was further cleaved by the cancer biomarker APN, and the catalytic efficiency of APN for CyA was calculated to be 0.03 μM. -1 s -1 ( Figure 9 (d and Table 1).

[0256] Example 3. TAMR's ability to detect TILs

[0257] The ability of the corresponding leukocyte test TAMR (prepared in Example 1) to detect TILs in the presence or absence of cancer biomarkers (APN, Pasqualini, R. et al., Cancer Res. 2000, 60, 722-727).

[0258] Cell viability test

[0259] 4T1 cells, CT26 cells, M1 macrophages, CD8 T cells, and MPRO cells were cultured at 8000 cells per well in 24-well plates for 24 h, followed by incubation with TAMR at a final concentration of 12.5–100 μg / mL for another 24 h. Cells from each well were collected, centrifuged to remove TAMR, and resuspended in MTS solution (200 μL, 0.1 mg / mL) and incubated at 37°C for an additional 4 h. The absorbance of the MTS solution was measured at 490 nm using a SpectraMax M5 microplate reader. Cell viability was calculated by comparing the absorbance of TAMR-incubated cells with that of the corresponding control cells.

[0260] Cellular imaging of TAMR

[0261] To perform cellular imaging for TAMR, 4T1 cells, CT26 cells, 3T3 cells, M1 macrophages, M2 macrophages, RAW264.7 cells, BMDCs, CD8 T cells, and MPRO cells were seeded into confocal cell culture dishes (5 × 10⁻⁶ cells / mL). 4 Cells / dish. After 24 h of incubation, cells were replaced with fresh medium containing TAMR (10 μM) in the absence or presence of APN (0.5 μg). After 2 h of incubation, cells were washed three times with PBS and then fixed with 4% paraformaldehyde (PFA) for 20 min. Cells were then stained with DAPI targeting the nucleus. Fluorescence images of cells were captured using an LSM800 (Zeiss). Fluorescence intensity was quantified using Image J.

[0262] Results and discussion

[0263] Note that all TAMRs showed negligible cytotoxicity against selective cells in TIME. Figure 10 When incubated with various leukocytes in the absence of APN, TAMRs exhibited negligible fluorescence signals. Only in the presence of APN did the NIR fluorescence of TAMRs activate in their target leukocytes, at a level 8 to 10 times higher than in non-specific leukocytes (Figs. 5g, h, and 11–15). These data further demonstrate that the fluorescence activation of TAMRs requires both cancer and leukocyte biomarkers, thus ensuring their specificity for TILs.

[0264] Example 4. Synthesis of AMR

[0265] (Cy-DAVY, Cy-DFEI, and Cy-VPAA) AMR precursors were synthesized according to a previous protocol (He, S. et al., J. Am. Chem. Soc. 2020, 142, 7075-7082). AMR was synthesized using the protocol for TAMR described in Example 1.

[0266] Cy-DAVY

[0267] Cy-DAVY MS: m / z 1062.61. 1 H NMR (400MHz, CDCl3): δ (ppm): 8.77 (d, J = 14, 1H), 7.76-7.71 (m, 3H), 7.58-7.55 (m, 2H), 7.50-7.41 (m, 4H), 7.39-7.33 (m, 1H), 7.06(d,J=8,4H),6.70(d,J=8,2H),6.53(d,J=12,1H),5.25(s,2H),4.76(t,1H),4.59-4.55(m,1H),4.36(t,2H),4.27-4.24(m ,1H),4.10(t,1H),3.46(t,2H),3.28-3.10(m,1H),3.05-3.0(m,1H),2.96(t,1H),2.91-2.85(m,2H),2.79-2.72(m,2H),2.74 -2.71(m,2H),2.08-2.03(m,2H),2.0-1.97(m,3H),1.91(s,3H),1.83(s,6H),1.71(m,1H),1.36(d,J=8,3H),1.02-0.9(m,6H).

[0268] Cy-DFEI

[0269] MS of Cy-DFEI: m / z 1118.58. For Cy-DFEI 1 1H NMR (400 MHz, CDCl3): δ (ppm): 8.76 (d, J = 16, 1H), 7.75 - 7.72 (m, 3H), 7.56 (d, J = 4, 2H), 7.50 - 7.47 (m, 4H), 7.41 (s, 1H), 7.23 - 7.16 (m, 4H), 7.12 - 7.07 (m, 3H), 6.53 (d, J = 16, 1H), 5.28 (s, 2H), 4.8 (t, 1H), 4.54 - 4.51 (m, 1H), 4.38 (t, 2H), 4.26 - 4.23 (m, 1H), 4.13 (d, J = 8, 1H), 3.48 (t, 2H), 3.37 (s, 2H), 3.20 - 3.15 (m, 1H), 3.06 - 2.96 (m, 2H), 2.79 - 2.73 (m, 5H), 2.34 - 2.23 (m, 2H), 2.04 (s, 3H), 1.97 - 1.93 (m, 4H), 1.84 - 1.76 (s, 6H), 1.46 - 1.4 (m, 3H), 1. 3 - 1.19 (m, 2H), 0.90 - 0.88 (m, 6H).

[0270] Cy-VPAA

[0271] 1 1H NMR (400 MHz, CDCl3): δ (ppm): 8.80 (d, J = 16, 1H), 7.75 - 7.73 (m, 2H), 7.64 - 7.62 (m, 2H), 7.57 - 7.52 (m, 2H), 7.49 - 7.43 (m, 2H), 7.35 - 7.27 (m, 2H), 7.13 - 7.06 (m, 2H), https: / / tosv.byted.org / obj / labcv-ocr / Seed_PDF_figures / figure_2024_4_11_1_33_31_46_174_131_377_231.jpg 6.55 (d, J = 16, 1H), 5.25 (s, 2H), 4.39 (t, 1), 4.29 (t, 3H), 4.23 - 4.17 (m, 2H), 3.83 - 3.81 (m, 2H), 3.67 - 3.62 (m, 2H), 3.54 - 3.46 (m, 2H), 3.24 - 3.15 (m, 1H), 2.82 (s, 2H), 2.75 (t, 1H), 2.72 (s, 2H), 2.68 - 2.65 (m, 2H), 2.53 (d, J = 8, 1H), 2.06 - 1.94 (m, 4H), 1.66 - 1.59 (m, 2H), 1.82 (s, 6H), 1.45 - 1.4 (m, 2H), 1.36 - 1.34 (m, 6H), 1.22 - 1.15 (m, 2H), 0.96 - 0.94 (m, 6H).

[0272] Example 5. In vivo specificity of TAMR for TIL

[0273] The development of imaging agents with high specificity for TILs remains challenging due to the presence of leukocytes in peripheral blood and inflamed tissues (Nourshargh, S. & Alon, R., Immunity 2014, 41, 694-707). The specificity of TAMR (prepared in Example 1) for TILs was investigated and compared with their monoclonal counterparts (AMR prepared in Example 4) (Figure 16a). Blood samples from mice treated with saline or LPS were incubated with these reporters for NIRF imaging (Figure 16b).

[0274] Absorption analysis

[0275] LPS (5.0 mg / kg) was injected intraperitoneally into live mice, and LPS-inflamed blood samples were collected 4 h post-injection. TAMR and AMR (10 μM) were incubated with LPS-inflamed blood samples and saline-treated blood samples for 30 min, respectively. The blood samples were then homogenized and centrifuged for 10 min. The supernatant was used for absorption analysis.

[0276] In vivo specific detection

[0277] For the blood incubation experiment, blood samples were first obtained from BALB / c mice. Briefly, saline or LPS (5 mg / kg mouse) was injected intraperitoneally into live mice. Blood samples were collected 4 h later. TAMR (final concentration: 10 μM) was added to the saline-treated blood sample and the LPS-inflamed blood sample (50 μL). Fluorescence images of the blood samples were obtained 30 min later using an IVIS spectral imaging system. Excitation: 675 nm. Emission: 720 nm.

[0278] For in vivo specificity detection, live mice were first constructed with LPS-inflamed tissue in the left thigh muscle and a subcutaneous CT26 tumor in the right flank. Briefly, the CT26 tumor was first inoculated into the right flank. Seven days later, LPS (5 mg / kg) was injected into the left thigh muscle. Twenty-four hours later, 10 μL of TAMR or AMR (0.1 mM) was locally injected into both the LPS-inflamed tissue and the CT26 tumor for longitudinal NIRF imaging. NIRF images were obtained using an IVIS spectral imaging system. Excitation: 675 nm. Emission: 720 nm.

[0279] Results and discussion

[0280] A fundamental challenge in molecular imaging of TILs lies in the lack of probe designs to distinguish TILs from resident leukocytes in other organs. Our dual-lock tandem molecular design addresses this challenge by incorporating both disease site and biomarker specificity into the probe's signal activation. The dual-lock TAMR triggers fluorescence only in the presence of both cancer and leukocyte biomarkers, and therefore, the specific detection of TILs in LPS-induced inflammation is free of false positives from other leukocytes; in contrast, their single-lock counterparts do not achieve this (Figure 16).

[0281] Compared to their pure forms in PBS solution, TAMRs exhibited almost identical NIRF signals in saline-treated blood samples, with a slight increase (1.1 to 1.2-fold) in LPS-inflamed blood samples. In contrast, AMRs showed NIRF signals in saline-treated and LPS-inflamed blood samples that were 1.8 to 3.6 times and 2.8 to 4.9 times higher, respectively, than their NIRF signals in PBS (Fig. 16c). The activation of AMRs was further confirmed by their higher absorption intensity at 680 nm in blood samples; this was 1.7 to 2.5 times higher than that of TAMRs. Figure 17 ).

[0282] The specificity of TAMR for TIL was further validated in live mice with LPS-inflamed tissue in the left thigh muscle (Ning, X. et al., Nat. Mater. 2011, 10, 602-607) and subcutaneous CT26 tumors in the right flank (Fig. 16d). Local injection of TAMR or AMR showed signal enhancement, which reached its maximum 30 min after injection of these reporters in both LPS-inflamed and tumor tissues (Fig. 18). Therefore, the reporter signals at 30 min after injection in both LPS-inflamed and CT26 tumor tissues were compared with the background signal of these reporters in the skin. Significant signal enhancement of TAMR was observed only in tumor tissue (3.6 to 4.2-fold), but not in LPS-inflamed tissue (1.1 to 1.6-fold); in contrast, significant signal enhancement of AMR was detected in both LPS-inflamed and tumor tissues (3.1 to 4.2-fold) (Fig. 16e, f). Additionally, when 4T1 or CT26 tumors were pretreated with the APN inhibitor bestatin (Shim, J Set al., Chem. Biol. 2003, 10, 695-704), the TAMR signal at the tumor site was slightly increased by 1.1 to 1.2 times compared to the reported skin background. Figure 19 This further demonstrates that cancer biomarkers are essential for TAMR activation.

[0283] Example 6. Clearance pathway and in vivo stability of TAMR

[0284] To determine the clearance pathway and in vivo stability of TAMR, urine was collected from healthy mice injected with TAMR (prepared in Example 1) or AMR (prepared in Example 4) and quantified by HPLC (Figure 16g).

[0285] Kidney clearance efficiency study

[0286] Intravenous injection of TAMR into healthy mice M1 TAMR CTL or TAMR NE (5 μmol / kg) and its monoclonal counterpart AMR M1 AMR CTL or AMR NE (5 μmol / kg) and placed in metabolic cages. Urine samples were collected at 3, 9, and 24 h post-injection. Renal clearance of these reporters was determined by HPLC analysis of the urine samples. The urine samples were also centrifuged at 5000 rpm for 15 min, and the UV-Vis absorption spectra of the supernatant were further recorded.

[0287] UV-Vis analysis of urine collected from healthy mice

[0288] Healthy mice were intravenously injected with either TAMR (5 μmol / kg) or AMR (5 μmol / kg) and placed in metabolic cages. Urine samples were collected at 12 time points after injection and centrifuged at 5000 rpm for 15 min. The UV-Vis absorption spectra of the supernatant were then recorded (n=3).

[0289] Results and discussion

[0290] Due to their high water solubility, these reporters exhibited renal clearance of 55-71% of the total injected dose at 24 h post-injection (Figs. 16h and 20). The signal intensity of TAMRs in excreted urine remained as low as their pure form in PBS, indicating high in vivo stability of TAMRs during systemic circulation and excretion. In contrast, the signal intensity of excreted AMRs was 1.6 to 1.8 times higher than that in PBS (Figs. 16i, j), and a novel absorption peak at 680 nm was observed for excreted AMRs assigned to the activated reporter. Figure 21 These data confirm that dual-lock TAMR minimizes nonspecific activation induced by circulating leukocytes, thus exhibiting high specificity for TILs.

[0291] Due to their high renal clearance, TAMRs can be applied to fluoroscopic urine analysis of time-dependent endothelial cells (TICs) to evaluate cancer immunotherapy, demonstrating significant clinical translational potential. In the following examples, we report the use of TAMRs for real-time NIRF imaging of triglycerides (TILs) for companion diagnostics and prediction of cancer immunotherapy.

[0292] Example 7. Real-time NIRF imaging of TILs in tumors with low immunogenicity

[0293] The ability of TAMR (prepared in Example 1) for in vivo real-time imaging of TILs was first validated in mice carrying 4T1 tumors, which were considered to be of low immunogenicity due to the presence of fewer tumor suppressor leukocytes (Taylor, MA et al., J. Immunother. Cancer 2019, 7, 328). Mice were intraperitoneally administered aPD-L1, Oxa, or a combination of aPD-L1 and Oxa (aPD-L1 / Oxa) (Figure 22a). Oxa, as a third-generation platinum drug, is known to inhibit cancer growth by inducing immunogenic cell death (ICD) that activates dendritic cells (DCs) and improves T cell infiltration (Zitvogel, L. et al., Nat. Rev. Immunol. 2008, 8, 59-73). Anti-programmed death ligand 1 (aPD-L1) has been used to inhibit the binding of PD-L136 (upregulated in solid tumors) to the receptor for programmed cell death protein 1 (PD-1) expressed on T cells for immune checkpoint inhibition (ICI) and to enhance adaptive immune responses against cancer cells.

[0294] Real-time in vivo imaging of TIL

[0295] One week after tumor inoculation, 4T1 tumor-bearing mice were intraperitoneally administered aPD-L1 (10 mg / kg), Oxa (6 mg / kg), or a combination of aPD-L1 and Oxa (aPD-L1 / Oxa) three times every two days. CT26 tumor-bearing mice were intraperitoneally administered aPD-L1 (10 mg / kg), aCD47 (10 mg / kg), or a combination of aPD-L1 and aCD47 (aPD-L1 / aCD47) three times every two days. On day 7, tumor penetration reference reporter PVP-IR800 (3 μmol / kg) and TAMR were administered intravenously. M1 TAMR CTL and TAMR NE The APN inhibition group received intravenous TAMR (5 μmol / kg) and was monitored with real-time NIRF imaging for 48 h. The APN inhibition group received intravenous TAMR. CTLTwo hours prior, betahistine (an APN inhibitor, 10 mg / mL, 10 μL) was injected intratumorally. Fluorescence imaging was performed at 675 nm excitation and 720 nm emission to monitor activated TAMR, and at 745 nm excitation and 800 nm emission to monitor IR800.

[0296] TIL in vitro real-time imaging

[0297] Mice were euthanized 72 h after injection of PVP-IR800 (3 μmol / kg) and TAMR (5 μmol / kg), and major tissues including heart, liver, spleen, lung, kidney and tumor were collected and captured using an IVIS spectral imaging system. Excitation at 675 nm and emission at 720 nm were used to monitor activated TAMR, and excitation at 745 nm and emission at 800 nm were used to monitor IR800.

[0298] After in vitro imaging, major organs were suspended in PBS, homogenized, and centrifuged (10,000 rpm, 10 min) to remove insoluble components. The distribution of TAMR was represented by analyzing the supernatant containing the extracted reporter by HPLC.

[0299] Immunofluorescence imaging

[0300] Mice were euthanized 24 h after TAMR (5 μmol / kg) injection, and tumors were collected and fixed in 4% PFA. After dehydration with 30% sucrose solution, tumor tissue was embedded in OCT medium for 10 min, followed by cryostating (Leica, CM1950) to 10 μm sections. Tumor sections were washed with PBS containing 0.1% Triton X-100 (PBST) and then incubated with 3% BSA solution at 25°C for 2 h to block non-specific antibody binding. Tumor sections were then subjected to TAMR-targeted therapies. M1 TAMR CTL and TAMR NE Alexa Group Staining was performed using 488 anti-F4 / 80 (C-7), PE anti-mouse CD8a, and NE polyclonal antibodies. For TAMR... NE The group further processed tumor sections using the second antibody Alexa. Conjugated goat anti-rabbit IgG was used for staining. Finally, tumor sections were stained with DAPI targeting the cell nucleus. Fluorescence images of the tumor sections were captured on an LSM800 (Zeiss). Finally, the colocalization of TAMRs with their corresponding NIRF signals from leukocytes was analyzed using ImageJ software.

[0301] In vitro flow cytometry analysis of white blood cells

[0302] Following three immunotherapy sessions, on day 8, 4T1 and CT26 tumor-bearing mice from each group were euthanized, and tumors, lymph nodes, and hematopoietic cells were harvested to prepare single-cell suspensions. For TIL evaluation, tumor tissue was cut into small pieces and digested for 4 h at 37°C in RPMI 1640 containing type I collagenase (1 mg / mL), type IV collagenase (100 μg / mL), and deoxyribonuclease I (100 μg / mL). The mixture was then filtered through a 70 μm cell filter. For leukocyte evaluation in lymph nodes and hematopoietic cells, lymph node cells and hematopoietic cells were treated with ACK lysis buffer to remove erythrocytes. All single-cell suspensions were first blocked with anti-mouse CD16 / 32, followed by live / dead staining. For CTL analysis, cells were incubated at 4°C using Alexa... Cells were stained with 700 anti-mouse CD45, FITC anti-mouse CD3, and PE anti-mouse CD8a for 30 min, followed by fixation with 4% PFA in the dark at 25°C for 20 min. Cells were then resuspended in ISPWB and incubated with APC anti-human / mouse GrB recombinant antibody at 25°C for 1 h, followed by washing three times with ISPWB. For M1 macrophage analysis, cells were incubated at 4°C using Alexa... 700 anti-mouse CD45, PerCP anti-mouse / human CD11b and Alexa Cells were stained with 488 anti-F4 / 80 (C-7) for 30 min, followed by fixation with 4% PFA in the dark at 25°C for 20 min. Afterward, cells were resuspended in ISPWB and fixed with PE anti-mouse NOS2 and Alexa at 25°C. Cells were incubated with 647 anti-Cas-1 (D-3) for 1 h, followed by three washes with ISPWB. For neutrophil analysis, cells were incubated at 4 °C using Alexa... Cells were stained with 700 anti-mouse CD45, PE anti-mouse / human CD11b, and APC anti-mouse Ly-6G for 30 min, followed by fixation with 4% PFA in the dark at 25°C for 20 min. Cells were then resuspended in ISPWB and incubated with NE polyclonal antibody at 25°C for 1 h, followed by washing three times with ISPWB. The cells were then treated with the secondary antibody Alexa. 488-conjugated goat anti-rabbit IgG was incubated at 25°C for 1 h, followed by three washes with ISPWB. The final cells were analyzed using a Fortessa X20 (BD Biosciences).

[0303] Results and discussion

[0304] Following three immunotherapy sessions, TAMR and the "always-on" reference reporter (PVP-IR800) were co-injected intravenously into 4T1 tumor-bearing mice for longitudinal NIRF imaging (Fig. 22a). NIRF signal intensity gradually increased in the tumor region, reaching a maximum at 24 h post-reporter injection (Fig. 22b), while the NIRF signal intensity of PVP-IR800 reached a maximum at 2 h post-reporter injection. Figure 23 Therefore, tumor signals were compared between different treatment groups at 24 hours post-reporter injection. To exclude the influence of TAMR concentration differences on the signal detected in tumors, a ratiometric signal of “TAMR” to “PVP-IR800” was defined and termed R-NIRF. M1 R-NIRF CTL or R-NIRF NE R-NIRF M1 The highest levels of R-NIRF were observed in mice treated with aPD-L1 / Oxa, specifically in aPD-L1-treated mice, Oxa-treated mice, and untreated mice. M1 1.1, 1.2, and 1.5 times higher. R-NIRF CTL The highest levels of R-NIRF were also observed in mice treated with aPD-L1 / Oxa, specifically in aPD-L1-treated mice, Oxa-treated mice, and untreated mice. CTL It is 1.4, 1.5, and 1.6 times higher. In comparison, R-NIRF... NE The R-NIRF levels were lowest in mice treated with aPD-L1 / Oxa, compared to those in mice treated with aPD-L1, mice treated with Oxa, and untreated mice. NE Treatment doses as low as 1 / 1.1, 1 / 1.1, and 1 / 1.2. These data indicate that TAMR [effects / conditions] in tumors of mice treated with aPD-L1 / Oxa [are effective / effective]. M1 and TAMR CTL It showed the highest activation activity, while TAMR NE It shows the lowest activation level.

[0305] Immunofluorescence staining showed that the red signal of TAMR overlapped well with the green signal of TIL labeled with FITC-labeled antibody (over 75%). Figure 27-29 Furthermore, double-positive cells, defined by those labeled with both TAMR and antibody, accounted for approximately 55% of the corresponding TILs (Figs. 22d and 27-29). Flow cytometry (Fig. 22e) revealed the presence of iNOS. + Cas-1 +The levels of iNOS cells were highest in tumors of mice treated with aPD-L1 / Oxa, and were the highest in aPD-L1-treated mice, Oxa-treated mice, and untreated mice, respectively. + Cas-1 + The levels at the cellular level were 1.9, 2.3, and 3.2 times higher. CD8 was found. + GrB + The levels of CD8 cells were highest in tumors of mice treated with aPD-L1 / Oxa, specifically in aPD-L1-treated mice, Oxa-treated mice, and untreated mice. + GrB + The levels in cells were 1.6-fold, 1.6-fold, and 3.4-fold higher. In contrast, Ly-6G was detected. + NE + The level of Ly-6G cells was lowest in the tumors of mice treated with aPD-L1 / Oxa, which was significantly lower than that in aPD-L1-treated mice, Oxa-treated mice, and untreated mice. + NE + Cell levels were as low as 1 / 1.9, 1 / 1.8, and 1 / 2.9. Similar trends in leukocytes were also observed in lymph nodes and peripheral blood (Figures 30-32). These data confirm that the activation sites of TAMR are specific to TILs, and that their signals correlate well with flow cytometry.

[0306] The relationship between TAMR and TIL was further evaluated using a simple linear regression model. R-NIRF was found. M1 and iNOS + Cas-1 + A positive correlation was found between cellular levels, with a correlation coefficient (R) of 0.75 and a Pearson R-value (ρ) of 0.82. R-NIRF CTL and R-NIRF NE respectively with CD8 + GrB + Cells and Ly-6G + NE + The cellular level showed a positive correlation (R CTL =0.86, ρ CTL =0.93, R NE =0.90 and ρ NE =0.95)(Figure 22f). Additionally, with CD45 +The proportions of cells (all white blood cells) further characterized TILs, showing that the aPD-L1 / Oxa-treated group had the largest population of CTLs (5.3%) and M1 macrophages (20.0%), but the smallest population of neutrophils (15.6%) (Fig. 22g). Furthermore, the R-NIRF proportions were in excellent agreement with the corresponding TIL proportions (R>0.70, p>0.83). Figure 33 These data confirm the effectiveness of ratiometric imaging signal (R-NIRF) for non-invasive and real-time characterization of TILs in 4T1 tumors.

[0307] Example 8. Real-time NIRF imaging of TILs in highly immunogenic tumors

[0308] The ability of TAMR (prepared in Example 1) to perform real-time in vivo imaging of TILs was further evaluated in mice carrying a CT26 tumor model, which was considered highly immunogenic due to the presence of a large number of tumor suppressor leukocytes (Taylor, MA et al., J. Immunother. Cancer 2019, 7, 328). Mice were intraperitoneally administered aPD-L1, antidifferentiation group 47 (aCD47), or a combination of aPD-L1 and aCD47 (aPD-L1 / aCD47), followed by systemic administration of TAMR and PVP-IR800 (Figure 34a). aCD47 has been widely used to block the interaction between CD47 on the tumor surface and the signal regulatory protein-α (SIRPα) overexpressed on the membranes of myeloid cells (including macrophages, DCs, etc.) (vanden Berg, TK & Valerius, T., Nature reviews. Clin. Oncol. 2019, 16, 275-276). Biological studies were conducted according to the protocol in Example 7.

[0309] Results and discussion

[0310] Following three immunotherapy sessions, TAMR and an "always-on" reference probe (PVP-IR800) were co-injected intravenously into live mice for longitudinal NIRF imaging (Fig. 34a). NIRF signals from TAMR and PVP-IR800 in the tumor region reached their maximum values ​​at 24 h and 2 h post-reporter injection, respectively (Figs. 34b and 35). Therefore, tumor R-NIRF was compared between different treatment groups at 24 h post-reporter injection. R-NIRFM1 was highest in mice treated with aPD-L1 / aCD47, being 1.3, 1.1, and 1.5 times higher than those in aPD-L1-treated, aCD47-treated, and untreated mice, respectively. CTLThe highest levels of R-NIRF were observed in mice treated with aPD-L1 / aCD47, and were found in mice treated with aPD-L1, mice treated with aCD47, and untreated mice. CTL It is 1.1, 1.4, and 1.9 times higher. In comparison, R-NIRF... NE The R-NIRF levels were lowest in mice treated with aPD-L1 / aCD47, compared to those in mice treated with aPD-L1, mice treated with aCD47, and untreated mice. NE As low as 1 / 1.3, 1 / 1.2, and 1 / 1.9 (Figures 34c and 36-38). These data suggest that TAMR [effects / effects] in tumors of mice treated with aPD-L1 / aCD47 [are effective / effective]. M1 and TAMR CTL It showed the highest activation activity, while TAMR NE It shows the lowest activation level.

[0311] Immunofluorescence staining of tumor sections showed that the red signal of TAMR overlapped well with the green signal of TIL labeled with FITC-labeled antibody. Figures 39-41 Double-positive cells accounted for 80-92% of TAMR-localized cells and 70-91% of the corresponding TILs (Figs. 34d and 39-41). Flow cytometry analysis showed the presence of iNOS. + Cas-1 + The levels of iNOS cells were highest in tumors of mice treated with aPD-L1 / aCD47, and were the highest in aPD-L1-treated mice, aCD47-treated mice, and untreated mice, respectively. + Cas-1 + The levels of CD8 were 1.6, 1.3, and 1.7 times higher at different cellular levels. + GrB + The level of CD8 cells was highest in tumors of mice treated with aPD-L1 / aCD47, and was respectively the highest in aPD-L1-treated mice, aCD47-treated mice, and untreated mice. + GrB + The levels of Ly-6G were 1.1-fold, 1.1-fold, and 1.3-fold higher at the cellular level, while Ly-6G was observed. + NE + The level of Ly-6G cells was lowest in the tumors of mice treated with aPD-L1 / aCD47, which was significantly lower than that in aPD-L1-treated mice, aCD47-treated mice, and untreated mice. + NE +Cell levels were as low as 1 / 1.6, 1 / 2.3, and 1 / 3.0. Leukocytes in lymph nodes and peripheral blood showed similar trends (Figures 42-44). These data confirm that the activation sites of TAMR are specific to TILs, and that their signals correlate well with flow cytometry.

[0312] The relationship between TAMR and TIL was further evaluated using a simple linear regression model. R-NIRF M1 iNOS in the tumor region + Cas-1 + The cellular level showed a positive correlation (R M1 =0.84, ρ M1 =0.91). R-NIRF CTL and R-NIRF NE Also with CD8 + GrB + Cells and Ly-6G + NE + The cellular level showed a positive correlation (R CTL =0.85, ρ CTL =0.92, R NE =0.88, ρ NE =0.94)(Figure 34f). Additionally, with CD45 + Cell proportions further characterized TILs, showing that the aPD-L1 / aCD47-treated group had the largest population of CTLs (21.8%) and M1 macrophages (26.5%), but the smallest population of neutrophils (8.9%) (Fig. 34g). Furthermore, a positive correlation was observed between R-NIRF and TIL proportions ( Figure 45 These results further confirm the effectiveness of ratiometric imaging signal (R-NIRF) for non-invasive and real-time characterization of tumor endothelial cells (TILs) in CT26 tumors.

[0313] Therefore, this array of TAMRs enables real-time multiplex characterization of TILs in TIME, providing a non-invasive method for accurately mapping the tumor immune context. Furthermore, TAMRs exhibit high specificity and sensitivity for TILs, showing over 70% overlap with their corresponding TILs in immunofluorescence staining of tumor sections (Figs. 22d and 34d), and their R-NIRF (R>0.74, P>0.81) are highly correlated with TIL levels measured by flow cytometry (Figs. 22f and 34f).

[0314] Example 9. Prediction of Cancer Immunotherapy

[0315] The efficacy of various treatments was evaluated in 4T1 and CT26 tumor-bearing mice.

[0316] Urine analysis of tumor-bearing mice

[0317] For urine analysis, 4T1 and CT26 tumor-bearing mice treated with different immunotherapies were intravenously injected with PVP-IR800 (3 μmol / kg mouse) and TAMR (prepared in Example 1, 5 μmol / kg mouse) and placed in metabolic cages. Urine samples were collected at 12 time points post-injection and imaged using an IVIS spectral imaging system, where 675 nm excitation and 720 nm emission were used to monitor activated TAMR, and 745 nm excitation and 800 nm emission were used to monitor IR800.

[0318] Tumor control rate

[0319] The tumor control rate is calculated using the following formula:

[0320] Tumor growth rate (TGR%) = Vt / Vt0 × 100%,

[0321] Tumor control rate (%) = [(TGR)] C -TGR S ) / TGR C ]×100%,

[0322] Vt0 refers to the tumor volume on day 0, Vt refers to the volume at a certain time, c refers to the control group (saline, unirradiated), and s refers to the group to be calculated.

[0323] Whole-slice imaging analysis of TAMR distribution

[0324] TAMR (prepared in Example 1, 5 μmol / kg) was intravenously injected into both 4T1 and CT26 tumor-bearing mice treated with different therapies. Twenty-four hours post-injection, the mice were euthanized, and the tumors were collected and fixed in 4% PFA. After dehydration with 30% sucrose, the tumor tissue was embedded in OCT medium and sectioned into 10-μm sections. The tumor sections were washed with PBST, followed by nucleus staining with DAPI. Fluorescent images of the tumor sections were captured on an LSM800 (Zeiss) using tiles mode.

[0325] ROC

[0326] We input in vivo fluorescence imaging data (R-NIRF) at 24h (n=3) and 48h (n=3) for each group into a GraphPad data table and plotted the data using a ROC curve model in the column analysis of GraphPad. Combinations of multiple TAMRs were derived from TAMRs. M1 TAMR CTL and TAMR NELogistic regression of R-NIRF.

[0327] PCA

[0328] We input the in vivo fluorescence imaging data of each group at 24h (n=3) and 48h (n=3) and the urine data at 12h (n=3) into the Origin workbook and created PCA plots using the enhanced version of Origin's principal component analysis tool.

[0329] Results and discussion

[0330] In 4T1 tumor-bearing mice, the highest tumor control rate (TCR) (85%) was observed with aPD-L1 / Oxa treatment, which was 9.9-fold and 1.8-fold higher than that of aPD-L1 treatment (8.6%) and Oxa treatment (48%), respectively (Figs. 46c and 47). Furthermore, the survival rate of mice treated with aPD-L1 / Oxa reached 83%, significantly higher than that of aPD-L1 (17%) and Oxa (50%). Note that all untreated mice died on day 28 (Fig. 46d). The high therapeutic efficacy of aPD-L1 / Oxa treatment is attributed to a synergistic effect: Oxa induces ICD generation and promotes antigen presentation to awakened CTLs. After CTL recruitment to TIME, aPD-L1 inhibits the binding of PD-L1 to PD-1 and promotes CTL cytotoxicity against cancer cells. Simultaneously, activated CTLs generated cytotoxic cytokines, which further promoted M1 macrophage polarization and suppressed immunosuppressive resident neutrophils, leading to effective killing of cancer cells (Fig. 46a). In CT26 tumor-bearing mice, the highest TCR (82%) was observed with aPD-L1 / aCD47 treatment, which was 1.2-fold and 1.3-fold higher than the TCRs of aPD-L1 treatment (71%) and aCD47 treatment (62%), respectively (Figs. 46e and 47). Mice treated with aPD-L1 / aCD47 achieved 100% survival, higher than aPD-L1 (83%) and aCD47 (50%). Note that all untreated mice died on day 32 (Fig. 46f). The high therapeutic efficacy of aPD-L1 / aCD47 treatment is attributed to a synergistic effect: aCD47 inhibits CD47-SIRPα interaction and promotes macrophage phagocytosis of apoptotic cells, which further enhances the initiation of CTLs. Meanwhile, aPD-L1 promotes the cytotoxicity of CTLs and provides an immunostimulatory microenvironment that inhibits the function of immunosuppressive neutrophils, thereby enhancing the anticancer efficacy (Figure 46b).

[0331] The high renal clearance efficiency and fluorescence-on-response of TAMR provide a convenient method for fluorescent urine analysis of TILs, making TAMR very promising for clinical translation. To evaluate the potential of TAMR for urine analysis, urine samples from mice after injection of the reporter agent were collected for NIRF measurement. Overall, the urine signal was in good agreement with real-time imaging data: for 4T1 tumor-bearing mice treated with aPD-L1 / Oxa, the urinary R-NIRF was [value missing]. M1 and R-NIRF CTL The highest level was observed in urinary R-NIRFNE, while the lowest was observed in CT26 tumor-bearing mice treated with aPD-L1 / aCD47. M1 and R-NIRF CTL It is the highest, while R-NIRF NE It is the lowest. (Figures 46g and 48-49). All urinary R-NIRF accurately reflect their corresponding TIL populations ( Figure 50 Consistent with real-time imaging data, the urinary R-NIRF of excreted TAMRs accurately reflected their corresponding TIL populations (R>0.74, ρ>0.85). Figure 50 ).

[0332] To determine whether urinary R-NIRF on day 7 could predict treatment outcome on day 20, a correlation analysis was performed between R-NIRF and relative tumor volume. This was consistent with in vivo ratioographic imaging data. Figure 51 For 4T1 and CT26 tumor-bearing mice, urinary R-NIRF M1 It showed a negative correlation with relative tumor volume, with correlation coefficients (R) of 0.91 and 0.79, and Pearson R values ​​(ρ) of -0.95 and -0.89, respectively. Additionally, urinary R-NIRF... CTL It is negatively correlated with relative tumor volume (4T1 model: R) CTL =0.85, ρ CTL = -0.92; CT26 model: R CTL =0.73, ρ CTL = -0.86 (Figure 46h). In contrast, urinary R-NIRFNE was positively correlated with relative tumor volume (4T1 model: R NE =0.92, ρ NE =0.96; CT26 model: R NE =0.89, ρ NE=0.94). This indicates that M1 macrophages and CTLs are positive prognostic indicators, while neutrophils are negative prognostic indicators of treatment outcome, consistent with previous clinical findings (Edin, S. et al., PLOS ONE 2012, 7, e47045; Galon, J. et al., Science 2006, 313, 1960-1964; and Kaneko, M. et al., Oncology 2012, 82, 261-268). Because urine analysis was performed on day 7, two weeks before the treatment endpoint, urinary R-NIRF for TAMR is a non-invasive and accurate method for predicting treatment outcome. Furthermore, comparisons of urinary R-NIRF for different tumors revealed a positive prognostic outcome in CT26 tumors with TAMR. M1 and TAMR CTL The presented R-NIRF values ​​are positive prognostic TAMR values ​​in 4T1 tumors. M1 and TAMR CTL The R-NIRF levels were 1.3 to 1.5 times higher. Therefore, TAMR M1 and TAMR CTL The tumors with low immunogenicity (4T1) and those with high immunogenicity (CT26) were clearly distinguished, thus demonstrating their ability to be stratified.

[0333] PCA was used to further evaluate the ability of TAMR in companion diagnostics and prediction of cancer immunotherapy. PCA of TAMR with R-NIRF clearly distinguished untreated 4T1 and CT26 tumors (Figure 46i). ROC analysis of TAMR for different tumor models revealed the potential of TAMR. M1 TAMR CTL and TAMR NE The AUCs were 0.92, 0.98, and 0.53, respectively, while the combination of TAMR better distinguished 4T1 and CT26 tumors (AUC = 1.00) (Figure 46j). The PCA of TAMR's R-NIRF also significantly differentiated aPD-L1-treated 4T1 and CT26 tumors (Figure 46k). ROC analysis of TAMR for aPD-L1-treated 4T1 and CT26 tumors revealed the benefits of TAMR. M1 TAMR CTL and TAMR NEThe AUCs were 0.81, 0.93, and 0.96, respectively, while the combination of TAMR better distinguished between aPD-L1-treated 4T1 and aPD-L1-treated CT26 tumors (AUC = 1.00) (Figure 46l). This result is consistent with previous data showing that CT26 tumors exhibit a strong response to ICI, while 4T1 tumors show little response to ICI (Mosely, SI et al., Cancer Immunol. Res. 2017, 5, 29-41). Therefore, TAMR-based multiplex urine analysis allows for the differentiation of untreated CT26 tumors and untreated 4T1 tumors, as well as aPD-L1-treated CT26 tumors and aPD-L1-treated 4T1 tumors, with high accuracy (AUC = 1.00) (Figure 46i-l). Furthermore, urinary R-NIRF of TAMR in early stages of cancer immunotherapy predicted the relative tumor volume of the endpoint, where TAMR M1 and TAMR CTL It showed a negative correlation (R>0.72, |ρ|>0.85), and TAMR NE Positive correlation was observed with relative tumor volume (R>0.88, ρ>0.93) (Fig. 46h). PCA of TAMR further significantly differentiated tumor-bearing mice in different treatment groups (Fig. 46m). Additionally, ROC analysis of multiple TAMR combinations showed high accuracy (AUC). 4T1 =0.98, AUC CT26 =1.00) distinguishes between untreated tumors and treated tumors ( Figure 52 These data demonstrate the potential of TAMR in patient stratification and evaluation of immunotherapy outcomes.

[0334] TAMR can also be used for microscopic examination of whole tumor sections, which is one of the clinical methods for evaluating TIL (Figure 46n, o). TAMR was observed in 4T1 tumor sections at their initial positions relative to the periphery of the untreated section. M1 and TAMR CTL The aPD-L1 / Oxa-treated slices diffused at densities 2 and 1.8 times higher, respectively. Figure 53 However, compared to untreated sections, TAMR was left at a density as low as 1 / 4.5 times at the periphery of sections treated with aPD-L1 / Oxa. NE For CT26 tumor sections, TAMR was observed compared to untreated tumor sections. M1 and TAMR CTL The aPD-L1 / aCD47-treated sections were non-uniformly distributed at densities 1.5 and 1.7 times higher, respectively; however, TAMR with densities as low as 1 / 3.1 remained in the aPD-L1 / aCD47-treated tumor sections.NE Because over 75% of TAMRs co-localize with TILs, as confirmed by immunofluorescence staining in Example 7 (Figures 22d and 34d), TAMRs in tumors represent the full picture of their corresponding tTILs. Furthermore, untreated 4T1 and CT26 tumors exhibit completely different immunospatial signatures, consistent with previous findings (Taylor, MA et al., J. Immunother. Cancer 2019, 7, 328), where highly immunogenic (CT26) tumors have high tumor suppressor leukocyte infiltration, while low immunogenic (4T1) tumors have low tumor suppressor leukocyte infiltration. Therefore, TAMRs allow for accurate differentiation between low-immunogenic (4T1) and high-immunogenic (CT26) tumors, and close monitoring of CTL infiltration, macrophage polarization, and neutrophil variation, along with different therapies used to predict treatment outcomes.

[0335] Therefore, in addition to urinalysis, TAMR can also depict the spatial distribution of TILs in whole tumor sections via microscopic examination, serving as another clinical application for the stratification and evaluation of cancer therapy. TAMR staining reveals changes in the location and density of TILs after therapy, showing a greater distribution of positive prognostic TILs (M1 macrophages and CTLs) and fewer negative prognostic TILs (neutrophils) in the center of the tumor, and that compared to untreated tumors, M1 macrophage levels were 1.5 to 2.0 times higher, CTL levels were 1.6 to 1.9 times higher, and neutrophil levels were as low as 1 / 3.1 to 1 / 4.5 (Fig. 46n, o).

[0336] In summary, we have developed an unprecedented suite of TIL-specific molecular fluorescent reporters (TAMRs) for companion diagnostics and prognosis in cancer immunotherapy. TAMRs possess a unique dual-locking sensing mechanism, enabling specific fluorescence association with TILs. Real-time imaging and urinalysis based on TAMRs are non-invasive and dynamic, yet capable of characterizing multiple TILs with the same sensitivity and specificity as static flow cytometry analysis and invasive biopsy. The signal correlation of TAMRs allows for precise analysis of tumor immunogenicity and longitudinal monitoring of TIME changes. Therefore, TAMRs not only provide a high-throughput, non-invasive, and efficient method for screening combination immunotherapy agents in a preclinical setting, but also have the potential to personalize patient stratification for combination cancer immunotherapy, optimize immunotherapy interventions, and predict immunotherapy outcomes in a clinical setting. The modular dual-locking tandem design of TAMRs can be extended to specifically detect biomarkers from target cells targeting disease sites, paving the way for precise biomarker characterization using molecular probes.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt or solvate thereof: in: X - Represents an anti-charge ion; and A represents the amino acid moiety that can be cleaved by enzymes associated with white blood cells.

2. The compound or its salt or solvate according to claim 1, wherein, A is selected from: The attached points are represented by dashed lines.

3. Use of the compound or its salt or solvate as defined in claim 1 or claim 2 in the preparation of an imaging agent for the diagnosis of lesions or diseases in tissues and / or organs using near-infrared fluorescence.

4. Use of the compound or its salt or solvation as defined in claim 1 or claim 2 in the preparation of an imaging agent for determining the susceptibility of tumor tissue to immunotherapy.

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