Photodynamic therapy probe for targeted degradation of PD-L1 and application of photodynamic therapy probe in tumor immunotherapy

Abstract

The invention discloses a photodynamic therapy probe for targeted degradation of PD-L1 and application of the photodynamic therapy probe in tumor immunotherapy. The probe is formed by covalent linkage of the following modules: a tumor targeted peptide: a peptide fragment with an amino acid sequence of CGNVVRQGC; the cell penetrating peptide is a peptide fragment of which the amino acid sequence is YGRKKRRQRRR; the PD-L1 degradation peptide is a peptide fragment with an amino acid sequence of MMDVKKC (Stbu) GIQDTNS and is used for competitively inhibiting PD-L1 palmitoylation, the Stbu is a GSH response module, the photosensitizer module is a TABY molecule, and alkynyl of the TABY molecule is connected with azide in the PD-L1 degradation peptide. The probe disclosed by the invention has the advantages of high tumor specificity, high PD-L1 degradation efficiency and high biocompatibility.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technologies, and particularly relates to a photodynamic therapy probe for targeted degradation of PD-L1 and its application in tumor immunotherapy. Background Art

[0002] Photodynamic therapy (PDT) is an innovative and non-invasive tumor treatment method that has shown great potential in clinical tumor treatment. In addition to directly killing tumor cells, PDT can also induce immunogenic cell death (ICD) in tumor cells. During the treatment process, damage-associated molecular patterns released by tumor cells can activate antigen-presenting cells. These antigen-presenting cells enhance the anti-tumor immune response by presenting antigens to T immune cells. However, although PDT can induce immunogenic death of tumor cells, it may also upregulate programmed death ligand 1 (PD-L1), which can lead to the formation of an immunosuppressive tumor microenvironment and become a key problem restricting the long-term efficacy of PDT. Therefore, it is urgent to systematically upgrade the PDT treatment system by improving the performance of photosensitizers and combining immunotherapy strategies.

[0003] Targeted protein degradation (TPD) technologies, including proteolysis-targeting chimeras (PROTACs) and autophagy-targeting chimeras (AUTACs), have become promising innovative strategies in the field of tumor immunotherapy. Compared with traditional small molecule inhibitors and antibodies, TPD shows unique advantages in dose regulation and targeting "undruggable" proteins, and can effectively overcome the drug resistance problems caused by existing small molecule inhibitors. At present, this technology has been successfully applied to the selective degradation of related proteins inside immune cells, on the surface of immune cell membranes, on tumor cell membranes, and inside tumor cells, thereby enhancing the anti-tumor immune response. Research shows that TPD synergistically remodels the tumor immunosuppressive microenvironment (TIME) through mechanisms such as downregulating immune checkpoint blockade proteins (such as PD-L1), degrading immune metabolism-related receptors, and inhibiting the expression of immune-related cytokines, and forms a synergistic therapeutic effect with the immune activation effect of photodynamic therapy (PDT). However, existing TPD strategies mostly lack the ability to specifically recognize disease-related cells, and their action mechanisms show a "continuous activation" characteristic, that is, regardless of the cell environment, once they contact the target protein, the degradation process is triggered. Given the key role of many proteins in maintaining normal cell function metabolism, this "always-on" TPD platform may have toxic side effects on normal cells in tumor treatment.

[0004] Therefore, in order to solve the above technical problems, it is necessary to develop a PD-L1 degradation strategy with strong specificity for tumor cells, which is of great significance for improving the efficacy of clinical PDT. Summary of the Invention

[0005] The object of the present invention is to provide a photodynamic therapy probe for targeted degradation of PD-L1 and its application in tumor immunotherapy. The probe of the present invention has the advantages of high tumor specificity, strong PD-L1 degradation efficiency and high biocompatibility.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, there is provided a photodynamic therapy probe for targeted degradation of PD-L1, which is composed of the following modules through covalent connection:

[0008] Tumor targeting peptide: a peptide segment with the amino acid sequence of CGNVVRQGC;

[0009] Cell penetrating peptide: a peptide segment with the amino acid sequence of YGRKKRRQRRR;

[0010] PD-L1 degradation peptide: a peptide segment with the amino acid sequence of MMDVKKC(Stbu)GIQDTNS, which is used for competitively inhibiting the palmitoylation of PD-L1, and the Stbu is a GSH-responsive group: N-succinimidyl-S-acetylthioacetate;

[0011] Photosensitizer module: a TABY molecule, and the alkyne group of the TABY molecule is connected to the azide in the PD-L1 degradation peptide;

[0012] The photodynamic therapy probe for targeted degradation of PD-L1 has the structure shown in formula (I):

[0013]

[0014] In the second aspect of the present invention, there is provided a preparation method of the photodynamic therapy probe for targeted degradation of PD-L1, and the method includes:

[0015] Mix the photosensitizer TABY, a copper catalyst, a polypeptide precursor XTP and SA, react in an organic solvent under the protection of an inert gas to obtain a reaction solution; wherein, the amino acid sequence of the polypeptide precursor XTP is: CGNVVRQGC-YGRKKRRQRRR-MMDVKKC(Stbu)GIQDTNS;

[0016] Separate, purify and dry the reaction solution to obtain the photodynamic therapy probe for targeted degradation of PD-L1.

[0017] Furthermore, the reaction conditions include: the temperature is 38-45 °C and the time is 10-14 hours.

[0018] Furthermore, the molar ratio of the photosensitizer TABY, copper catalyst, polypeptide precursor XTP, and SA is: (1.8 - 2.2) : (4.5 - 5.5) : 1 : (0.9 - 1.1).

[0019] Furthermore, the copper catalyst is selected from one of copper bromide and copper chloride.

[0020] Furthermore, the organic solvent is DMSO.

[0021] Furthermore, the SA is Sodium ascorbate, CAS number: 134 - 03 - 2, and in the reaction, SA plays a catalytic role when the polypeptide and TABY are linked.

[0022] Furthermore, the structural formula of the photosensitizer TABY in the embodiments of the present invention is as shown below, and its preparation method can be found in patent application CN 116947757 A.

[0023]

[0024] In the third aspect of the present invention, there is provided the use of the above-mentioned photodynamic therapy probe for targeted degradation of PD - L1 in the preparation of anti - tumor drugs.

[0025] Specifically applied to photodynamic therapy, after the photodynamic therapy probe for targeted degradation of PD - L1 is intravenously injected, a 660 nm laser (800 mW / cm 2 , 5 min) irradiates the tumor site to activate the photodynamic / photothermal effect and degrade PD - L1.

[0026] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0027] 1. A novel polypeptide - photosensitizer probe XTPA provided by the present invention realizes the specific degradation of PD - L1 in tumor cells through the following design:

[0028] (1) Tumor cell targeting module TMTP1: Targets the XPNPEP2 protein highly expressed on the membrane of highly metastatic tumor cells.

[0029] (2) Cell - penetrating module TAT: Can increase the uptake of the probe by cells, enabling the probe to achieve high enrichment inside cells.

[0030] (3) PD - L1 degradation module P: Competes with endogenous PD - L1 for palmitoylation, thereby reducing the palmitoylation of endogenous PD - L1 and increasing ubiquitination, resulting in the degradation of endogenous PD - L1.

[0031] (4) Photosensitizer TABY: Has excellent type I, type II photodynamic and mild photothermal properties.

[0032] 2. The novel polypeptide-photosensitizer probe XTPA provided by the present invention has excellent tumor targeting:

[0033] (1) High tumor targeting: Confocal images show that after incubating XTPA with normal cells (HLF cells) and tumor cells (A375 cells) for a certain period of time ( Figure 12 ), the fluorescence intensity in tumor cells is much higher than that in normal cells, indicating its excellent tumor targeting.

[0034] (2) Specific response and degradation in tumor cells: Compared with normal cells, the GSH concentration in tumor cells is much higher than that in normal cells. Mass spectrometry data show that XTPA can expose its active site to degrade PD-L1 only under high concentrations of GSH ( Figure 17 and Figure 18 ).

[0035] 3. The novel polypeptide-photosensitizer probe XTPA provided by the present invention can significantly degrade PD-L1 in tumor cells:

[0036] (1) High degradation efficiency of PD-L1: The degradation of PD-L1 in tumor cells by XTPA is concentration-dependent. Almost complete degradation of PD-L1 in A375 cells can be achieved at 10 μM ( Figure 16 ).

[0037] (2) XTPA can reverse the increase in PD-L1 caused by single photodynamic therapy in vivo and enhance the anti-tumor effect of photodynamic therapy ( Figure 19 , Figure 20 and Figure 21 ).

[0038] 4. The novel polypeptide-photosensitizer probe XTPA provided by the present invention has excellent biocompatibility:

[0039] (1) Low dark toxicity: MTT experiments confirm that without light irradiation, XTPA has almost no effect on the viability of tumor cells A375 cells ( Figure 15 ), indicating its excellent biocompatibility.

[0040] (2) No side effects: The body weight of mice and HE sections confirm that during the treatment with XTPA, the body weight of mice shows almost no change, and the tissue morphology of each organ shows no change, indicating that XTPA has almost no toxic side effects on the body ( Figure 22 and Figure 23 ). Description of the Drawings

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0042] Figure 1 : Structure of XTPA.

[0043] Figure 2 : Mass spectrometry characterization results of XTPA.

[0044] Figure 3 : Mass spectrometry characterization results of the control probe XTA.

[0045] Figure 4 : Mass spectrometry characterization results of the control probe TPA.

[0046] Figure 5 : Mass spectrometry characterization results of the control probe XPA.

[0047] Figure 6 : Mass spectrometry characterization results of the control probe XTPA-Fitc.

[0048] Figure 7 : UV and fluorescence characterization results of XTPA and its control probes.

[0049] Figure 8 : TEM characterization of XTPA and its control probes.

[0050] Figure 9 : DLS characterization of XTPA and its control probes.

[0051] Figure 10 : Photothermal properties of XTPA and its control probes in solution.

[0052] Figure 11 : Photodynamic properties of XTPA and its control probes in solution.

[0053] Figure 12 : XTPA has good targeting to tumors.

[0054] Figure 13 : Photothermal properties of XTPA and its control probes in cells.

[0055] Figure 14 : Photodynamic properties of XTPA and its control probes in cells.

[0056] Figure 15 : Killing effect of XTPA and its control probes on A375 cells.

[0057] Figure 16 : Degradation effect of XTPA on PD-L1 in tumor cells.

[0058] Figure 17 : Responsiveness of XTPA to high concentration of GSH.

[0059] Figure 18 : XTPA can specifically degrade PD-L1 in tumor cells.

[0060] Figure 19 : Immunocytotoxicity effect of XTPA and its control probe on A375 cells.

[0061] Figure 20 : Antitumor effect of XTPA and its control probe on human immune A375 subcutaneous tumor model. Among them, Figure A is the experimental procedure, Figure B is the comparison result of tumor pictures of each group, and Figure C is the statistical result of the tumor inhibition rate of XTPA + Laser group and other light irradiation groups.

[0062] Figure 21 : Antitumor mechanism of XTPA and its control probe.

[0063] Figure 22 : Weight change results of mice in each group during the treatment with XTPA and its control probe.

[0064] Figure 23 : HE staining results of organs during the treatment with XTPA and its control probe. Detailed implementation manners

[0065] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than to limit the present invention.

[0066] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail. Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be obtained by existing methods.

[0067] The general idea of the present invention is as follows:

[0068] According to a typical implementation manner of the present invention, a photodynamic therapy probe for targeted degradation of PD-L1 is provided. The photodynamic therapy probe for targeted degradation of PD-L1 is composed of the following modules through covalent connection (see attachedFigure 1 ):

[0069] Table 1

[0070]

[0071] The application mechanism of the described photo-dynamic therapy probe targeting the degradation of PD-L1 in the preparation of anti-tumor drugs includes:

[0072] (1) GSH response: GSH (2 - 10 mM) in tumor cells reduces the acetylthioacetate group (-SCOCH3) of SA

[0073] (2) When the concentration of glutathione (GSH) is ≥ 2 mM, SA undergoes deacetylation reaction, the disulfide bond breaks, and the active group is exposed for the degradation of PD-L1;

[0074] (3) PD-L1 degradation: The PD-L1 degradation peptide and endogenous PD-L1 competitively bind to the DHHC3 enzyme, reducing the palmitoylation of endogenous PD-L1 and resulting in the ubiquitination and degradation of endogenous PD-L1.

[0075] Next, a photo-dynamic therapy probe targeting the degradation of PD-L1 of the present application and its application in tumor immunotherapy will be described in detail in combination with examples and experimental data.

[0076] Example 1: Synthesis and structural characterization of the photo-dynamic therapy probe targeting the degradation of PD-L1

[0077] 1. Preparation method:

[0078]

[0079] Dissolve TABY (2.93 mg, 4.51 μmol, 2 equiv.) and CuBr (1.62 mg, 11.27 μmol, 5 equiv.) in 1 mL of DMSO respectively, dissolve XTP (10.00 mg, 2.25 μmol, 1 equiv.) and SA (0.45 mg, 2.25 μmol, 1 equiv.) in 1 mL of DEPC water respectively. Mix the above four substances in sequence and stir the reaction overnight under N2 protection at 42 °C. After the reaction, separate and purify by HPLC, and then freeze-dry with a vacuum dryer to obtain a blue solid. The polypeptide precursor XTP of the present invention example is synthesized by the company, and the amino acid sequence of the polypeptide precursor XTP is:

[0080] CGNVVRQGC-YGRKKRRQRRR-MMDVKKC(Stbu)GIQDTNS.

[0081] II. Characterization results:

[0082] The mass spectrometry results are as follows Figure 2 shown: indicating that XTPA was successfully synthesized.

[0083] Comparative Example 1, control probe XTA

[0084]

[0085] Dissolve TABY (4.65 mg, 7.15 μmol, 2 equiv.) and CuBr (2.56 mg, 17.87 μmol, 5 equiv.) separately in 1 mL of DMSO, dissolve XT (10.00 mg, 3.57 μmol, 1 equiv.) and SA (0.71 mg, 3.57 μmol, 1 equiv.) separately in 1 mL of DEPC water. Mix the above four substances in sequence and stir the reaction overnight under N2 protection at 42 °C. After the reaction is completed, separate and purify by HPLC, and then freeze-dry with a vacuum dryer to obtain a blue solid.

[0086] The mass spectrometry characterization of the control probe XTA is as follows Figure 3 shown, indicating that it was successfully synthesized.

[0087] Comparative Example 2, control probe TPA

[0088]

[0089] Dissolve TABY (3.82 mg, 5.87 μmol, 2 equiv.) and CuBr (2.11 mg, 14.67 μmol, 5 equiv.) separately in 1 mL of DMSO, dissolve TP (10.00 mg, 2.93 μmol, 1 equiv) and SA (0.58 mg, 2.93 μmol, 1 equiv.) separately in 1 mL of DEPC water. Mix the above four substances in sequence and stir the reaction overnight under N2 protection at 42 °C. After the reaction is completed, separate and purify by HPLC, and then freeze-dry with a vacuum dryer to obtain a blue solid.

[0090] The mass spectrometry characterization of the control probe TPA is as follows Figure 4 shown, indicating that it was successfully synthesized.

[0091] Comparative Example 3, control probe XPA

[0092]

[0093] Dissolve TABY (4.49 mg, 6.91 μmol, 2 equiv.) and CuBr (2.48 mg, 17.27 μmol, 5 equiv.) separately in 1 mL of DMSO. Dissolve XP (10.00 mg, 3.45 μmol, 1 equiv.) and SA (0.68 mg, 3.45 μmol, 1 equiv.) separately in 1 mL of DEPC water. Mix the above four substances in sequence and stir the reaction overnight under N₂ protection at 42 °C. After the reaction, separate and purify by HPLC, and then freeze-dry with a vacuum dryer to obtain a blue solid.

[0094] The mass spectrometry characterization of the control probe XPA is as Figure 5 shown, indicating that it was successfully synthesized.

[0095] Comparative Example 4, control probe XTPA-Fitc

[0096]

[0097] Dissolve TABY (2.63 mg, 4.04 μmol, 2 equiv.) and CuBr (1.45 mg, 10.10 μmol, 5 equiv.) separately in 1 mL of DMSO. Dissolve X(F)TP (10.00 mg, 2.02 μmol, 1 equiv.) and SA (0.40 mg, 2.02 μmol, 1 equiv.) separately in 1 mL of DEPC water. Mix the above four substances in sequence and stir the reaction overnight under N₂ protection at 42 °C. After the reaction, separate and purify by HPLC, and then freeze-dry with a vacuum dryer to obtain a blue solid.

[0098] The mass spectrometry characterization of the control probe XTPA-Fitc is as Figure 6 shown, indicating that it was successfully synthesized.

[0099] Experimental Example 1, performance detection

[0100] 1. UV and fluorescence characterization of XTPA and its control probes

[0101] Prepare 10 μM of XTPA and its control probes and perform UV-visible absorption spectroscopy tests.

[0102] As Figure 7 shown in A, XTPA, XTA, TPA, XPA and TABY have similar characteristic absorption peaks at 600 nm; X(F)TPA contains TABY and FITC, so it has two characteristic absorption peaks at 480 nm and 600 nm, indicating that TABY was successfully linked to the polypeptide. As Figure 7As shown in Figure B, since the fluorescence intensity of the TABY molecule itself is not high, XTPA, XTA, TPA, and XPA do not show fluorescence, and X(F)TPA shows strong fluorescence signals at 520 nm due to the inclusion of FITC.

[0103] 2. TEM Characterization of XTPA and Its Control Probes

[0104] Drop the XTPA, XTA, TPA, and XPA probe solutions onto the copper grid, and observe the morphology after negative staining through a transmission electron microscope (TEM).

[0105] The results are as Figure 8 shown. The TEM results show that the self-assembled nanostructures formed by XTPA have a particle size of 60 nm, XTA and TPA are about 120 nm, and XPA reaches 180 nm. Each group of probes has a good particle size distribution.

[0106] 3. DLS Characterization of XTPA and Its Control Probes

[0107] Measure the hydrodynamic particle size of XTPA, XTA, TPA, and XPA using a dynamic light scattering instrument (DLS).

[0108] The results are as Figure 9 shown. The DLS results show that XTPA has a hydrodynamic particle size of 90.24 nm, XTA and TPA are 107.9 and 137.2 nm respectively, and XPA reaches 214.4 nm. Each group of probes has a good particle size distribution.

[0109] 4. Photothermal Properties of XTPA and Its Control Probes in Solution

[0110] Take 100 μM probe solution, irradiate it with a 660 nm laser, and record the temperature change with an infrared thermal imager. The results are as Figure 10 shown. XTPA, XTA, TPA, and XPA all exhibit good photothermal effects. 5. Photodynamic Properties of XTPA and Its Control Probes in Solution

[0111] Mix 10 μM probe solution with ABDA probe, irradiate it with a 660 nm laser, and monitor the decrease rate of absorbance at 412 nm

[0112] (ROS oxidizes ABDA).

[0113] The results are as Figure 11 shown. XTPA and XTA have good photodynamic properties.

[0114] 6. XTPA Has Good Targeting to Tumors

[0115] X(F)TPA (fluorescently labeled probe) was incubated with HLF cells (normal) and A375 cells (tumor) respectively, and the fluorescence intensity was observed by confocal microscopy.

[0116] The results were as Figure 12 shown. Compared with HLF cells, A375 cells had higher expression of XPNPEP2. After incubating HLF and A375 cells with X(F)TPA, the confocal results showed that the fluorescence intensity of A375 cells was higher, about 1.5 times that of HLF cells, indicating that X(F)TPA could better target tumor cells.

[0117] 7. Photothermal properties of XTPA and its control probes in cells

[0118] A375 cells were treated with PBS, XTPA, XTA, TPA, XPA (10 μM) for 24 h respectively, irradiated with 660 nm laser, and the cell temperature was recorded by infrared thermal imaging.

[0119] The results were as Figure 13 shown. XTPA could increase the temperature of A375 cells by about 20 °C.

[0120] 8. Photodynamic properties of XTPA and its control probes in cells

[0121] A375 cells were treated with PBS, XTPA, XTA, TPA, XPA (10 μM) for 24 hours respectively, then treated with DCFH-DA for 20 min, irradiated with 660 nm laser for 5 min, and the green fluorescence was detected by confocal microscopy.

[0122] The results were as Figure 14 shown. In the presence of ROS, non-fluorescent DCFH could be oxidized to fluorescent DCF. Confocal images showed that more ROS were generated in cells treated with XTPA and XTA after laser irradiation, with the most in XTPA, while less ROS were generated in TPA and XPA.

[0123] 9. Killing effect of XTPA and its control probes on A375 cells

[0124] After A375 cells were treated with PBS, XTPA, XTA, TPA, XPA (10 μM) for 24 hours respectively, before and after light irradiation (power: 800 mW / cm 2 , time: 5 min), the viability of A375 cells was tested by MTT assay.

[0125] The results were as Figure 15 shown. After light irradiation, 60% of the cell viability of A375 cells was lost with XTPA.

[0126] 10. Effect of XTPA on the degradation of PD-L1 in tumor cells

[0127] A375 cells were incubated with different concentrations of XTPA for 24 hours, and PD-L1 protein expression in the cells was detected by Western blotting. XTPA showed a concentration-dependent effect on PD-L1 degradation, with higher concentrations of XTPA being more effective. A375 cells were incubated with XTPA, XTA, TPA, and XPA (10 μM) for 24 hours, and PD-L1 protein expression in the cells was detected by Western blotting.

[0128] The results are as follows Figure 16 As shown, XTPA significantly reduced PD-L1 expression, followed by XPA and TPA, while XTA had no effect. This is because the pPD-L1 peptide present in XTPA, XPA, and TPA can competitively inhibit endogenous PD-L1, thereby interfering with the palmitoylation process of PD-L1 in vivo and profoundly downregulating PD-L1 expression on the cell membrane. XPA, however, lacks the TAT peptide, which impairs cellular uptake of the probe. TPA, however, lacks the TMTP1 peptide, preventing it from specifically targeting highly metastatic tumor cells.

[0129] 11. XTPA responsiveness to high concentrations of GSH

[0130] After incubation of XTPA and 10 mM GSH for 24 h, the molecular weight changes were detected by high-resolution mass spectrometry.

[0131] The results are as follows Figure 17 As shown in Figure 3, in the presence of 10 mM GSH, XTPA can be deprotected, exposing the active site.

[0132] 12. XTPA can specifically degrade PD-L1 in tumor cells

[0133] A375 cells were treated with PBS, H2O2 and GSH (100 μM) for 24 hours, and PD-L1 was detected by WB.

[0134] The results are as follows Figure 18 As shown in the results, under the combined action of high concentrations of GSH and XTPA, the PD-L1 expression of A375 cells was significantly reduced, and the expression of PD-L1 was negatively correlated with the concentration of GSH.

[0135] 13. Immune killing effect of XTPA and its control probe on A375 cells

[0136] A375 cells after different treatments were co-incubated with PBMCs for 12 h. The killing of A375 cells by aPBMCs was observed using CLSM, and tumor cell death (red fluorescence weakened) was observed using confocal microscopy.

[0137] The results were as follows Figure 19 shown. A375 cells showed red fluorescence, and PBMCs showed green fluorescence. Confocal microscopy showed that the red fluorescence in the XTPA+Laser group was the weakest because XTP and XTPA could downregulate the expression of PD-L1 in A375 cells. Therefore, aPBMCs could better recognize and kill them. In the XTPA+Laser group, the superimposed effects of immunogenicity and downregulation of PD-L1 expression showed stronger immune activation and tumor killing effects.

[0138] Experimental Example 2: Anti-tumor experiment in vivo

[0139] 1. Anti-tumor effects of XTPA and its control probes on a human immune A375 subcutaneous tumor model

[0140] An A375 subcutaneous tumor mouse model was constructed by injecting A375 cells subcutaneously into immunodeficient NOG mice. When the tumor grew to approximately 200 mm 3 , human PBMCs were transplanted. After three treatments, the mice were dissected and subsequent tests were performed.

[0141] The results were as follows Figure 20 shown. Compared with the PBS group, the tumor inhibition rate in the XTPA group was 36%, while the tumor inhibition rate in the XTPA+Laser group was as high as 95%. The tumor inhibition rates in the other groups treated with phototherapy, namely XTA+Laser, TPA+Laser, and XPA+Laser, were 44%, 59%, and 61% respectively.

[0142] 2. Anti-tumor mechanisms of XTPA and its control probes

[0143] HE and PD-L1 immunofluorescence staining were performed on tumor tissues after different treatments. The HE results showed that more tumor cell deaths occurred in the XTPA+Laser group, manifested as more coagulation condensation, fragmentation, or anuclear cells.

[0144] The results were as follows Figure 21 shown. PD-L1 immunofluorescence staining showed that due to the lack of pPD-L1 peptide in XTA, after light stimulation, the expression of PD-L1 not only did not decrease but increased slightly in tumor cells. XTPA reduced the palmitoylation of endogenous PD-L1, resulting in an increase in its ubiquitination degree, and further increasing the degradation of PD-L1, thus reducing the expression of PD-L1 in tumor tissues.

[0145] 3. Changes in mouse body weight during treatment

[0146] The results were as follows Figure 22As shown, during the treatment process of XTPA and its control probe, there were no significant differences in the body weights of the mice in each group, indicating that the probe has good biosafety.

[0147] 4. In vivo biosafety analysis

[0148] The heart, liver, spleen, lungs, and kidneys were taken for H&E staining.

[0149] The results are as Figure 23 shown. During the treatment process of XTPA and its control probe, by performing HE staining on the organs, the results showed that there were no significant differences in the tissues of each group of organs, further indicating the biosafety of the probe.

[0150] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0151] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0152] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A photodynamic therapy probe for targeted degradation of PD-L1, characterized in that, The photodynamic therapy probe for targeted degradation of PD-L1 is composed of the following modules through covalent connection: Tumor targeting peptide: a peptide segment with the amino acid sequence CGNVVRQGC; Cell penetrating peptide: a peptide segment with the amino acid sequence YGRKKRRQRRR; PD-L1 degrading peptide: a peptide segment with the amino acid sequence MMDVKKC(Stbu)GIQDTNS, which is used for competitively inhibiting the palmitoylation of PD-L1, and the Stbu is a GSH-responsive group: N-succinimidyl-S-acetylthioacetate; Photosensitizer module: the TABY molecule, and the alkyne group of the TABY molecule is connected to the azide in the PD-L1 degrading peptide; The photodynamic therapy probe for targeted degradation of PD-L1 has the structure shown in formula (I); 2. A method for preparing a photodynamic therapy probe for targeted degradation of PD-L1 according to claim 1, characterized in that, The method includes: Reacting a photosensitizer TABY, a copper catalyst, a polypeptide precursor XTP, and SA in an organic solvent under the protection of an inert gas to obtain a reaction solution; wherein, the amino acid sequence of the polypeptide precursor XTP is: CGNVVRQGC-YGRKKRRQRRR-MMDVKKC(Stbu)GIQDTNS; Separating, purifying, and drying the reaction solution to obtain the photodynamic therapy probe for targeted degradation of PD-L1.

3. The method according to claim 2, wherein The reaction conditions include: the temperature is 38-45 °C, and the time is 10-14 hours.

4. The method according to claim 2, characterized in that, The molar ratio of the photosensitizer TABY, the copper catalyst, the polypeptide precursor XTP, and SA is: (1.8-2.2):(4.5-5.5):1:(0.9-1.1).

5. The method according to claim 2, characterized in that, The copper catalyst is selected from one of copper bromide and copper chloride.

6. Use of the photodynamic therapy probe for targeted degradation of PD-L1 according to claim 1 in the preparation of an anti-tumor drug.

7. An anti-tumor drug, characterized in that, Comprising the photodynamic therapy probe for targeted degradation of PD-L1 according to claim 1.

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