Design of enzyme-responsive in situ self-assembling peptide PSK and its anti-tumor use

Abstract

The application belongs to the technical field of biological materials, and particularly relates to an enzyme response in-situ self-assembled peptide and anti-tumor application thereof. An amino acid sequence of the self-assembled peptide PSK is shown in SEQ ID NO. 1. The self-assembled peptide PSK of the application firstly combines a PD-L1 blocking peptide with a tumor cell surface PD-L1, effectively internalizes the PSK peptide into the tumor cell through intracellular endocytosis, hydrolyzes in a lysosome through Legumain response bond, triggers self-assembled module assembly to form a beta-folded nanofilament structure, further triggers lysosome membrane permeability change, escapes from the lysosome, releases KLA to destroy mitochondria and induce tumor cell apoptosis. At the same time, the PD-L1 binding peptide is endocytosed into the lysosome, releases immunosuppression, combines with ICD induced by mitochondrial damage, enhances the killing effect of immune cells on tumor cells in the tumor microenvironment, effectively improves the selectivity and anti-tumor efficacy of the polypeptide, and has good biological safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological materials, and particularly relates to an enzyme-responsive in-situ self-assembled peptide and anti-tumor use thereof. BACKGROUND

[0002] Tumors pose a great threat to the physical and mental health and quality of life of global human beings, and conventional drugs have weak targeting ability and are easy to cause unnecessary damage to healthy tissues. At the same time, the development of tumor drug resistance can also seriously reduce the therapeutic effect of drugs. Targeted drug delivery treatment based on nanomaterials is considered to be one of the strategies with the greatest hope of overcoming the difficulties of cancer treatment. In recent years, many cancer treatment methods have emerged, among which targeted therapy and immunotherapy have become the main cancer treatment strategies. Although cancer treatment has made significant progress, the treatment effect and prognosis are still not satisfactory, and therefore more effective treatment strategies are urgently needed.

[0003] Immunotherapy that blocks immune checkpoints such as PD-L1 has been used for clinical treatment, but the limitation of insufficient immune stimulation is still a challenge for better efficacy. One way to overcome this limitation is to induce immunogenic cell death (ICD) by promoting tumor cells to release damage-associated molecular patterns (DAMPs) to enhance the anti-tumor immune response. This process can effectively convert immunologically "cold" tumors into "hot" tumors, making them more sensitive to immunotherapy.

[0004] Mitochondria are the main energy synthesis and regulation center of cells and play a key role in maintaining cell survival and function. Studies have shown that oxidative stress caused by mitochondrial dysfunction can trigger apoptosis autophagy of tumor cells. Tumor cells usually have abnormal mitochondrial morphology, number and function, which are closely related to their proliferation, survival and metastatic potential. These characteristics provide opportunities for targeted intervention and treatment strategies against mitochondrial dysfunction. Mitochondrial toxic peptide KLA is a pro-apoptotic peptide with an amphipathic alpha-helix conformation that can disrupt the integrity of the mitochondrial membrane, trigger the apoptosis program and cause cell death. However, the low internalization ability and lack of tumor cell specificity of KLA limit its anti-tumor efficacy. Therefore, effectively improving the internalization ability, permeability and accumulation of KLA in tumor cells is of great significance for enhancing the tumor killing ability of KLA. SUMMARY

[0005] In view of the oxidative stress caused by mitochondrial damage can induce ICD in tumor cells, and the anti-tumor specificity of mitochondrial toxic peptide KLA, the application provides a method of promoting ICD in tumor cells and blocking PD-L1 pathway to enhance anti-tumor immunity. The application provides an enzyme-responsive in situ self-assembled peptide to promote its targeted release and accumulation at the tumor site, significantly improving the safety and effectiveness of KLA. The application designs an enzyme-responsive self-assembled peptide PSK, which is composed of four functional components: a PD-L1 binding peptide, a legumain responsive module, a self-assembling module, and a mitochondrial toxic peptide KLA. The PD-L1 binding peptide promotes the binding of PD-L1 on the surface of tumor cells, promotes the effective uptake of PSK peptide through endocytosis, thereby improving the uptake and permeability of KLA. In addition, PD-L1 is endocytosed into lysosomes, relieving immunosuppression. The protease Legumain is overexpressed in various tumor tissues, but its expression in normal tissues is extremely low, and it can cut the PSK response site in lysosomes, triggering the self-assembling module to form a beta-sheet nanofiber structure. This further changes the permeability of the lysosomal membrane, allowing PSK to escape and enter mitochondria, leading to tumor cell apoptosis. At the same time, mitochondrial damage induces ICD, enhancing immune cell-mediated killing of tumor cells in the tumor microenvironment. PSK accumulates in tumor cells through receptor-specific binding, enzyme response, and self-assembly induction, thereby improving the selectivity and anti-tumor effect of the polypeptide. In summary, the application designs and synthesizes an in situ self-assembled peptide PSK targeting tumor cells and acting on mitochondria, which can induce apoptosis and ICD, effectively inhibit tumor development, and provide a new and reference method and idea for tumor treatment.

[0006] The application adopts the following technical solutions:

[0007] The application provides an enzyme-responsive in situ self-assembled peptide PSK under the action of enzymes in the tumor microenvironment, and the amino acid sequence of the self-assembled peptide PSK is KLAKLAKKLAKLAKKLVFFAANCVRARTR (SEQ ID NO. 1).

[0008] The application provides the use of the aforementioned enzyme-responsive in situ self-assembled peptide PSK in the preparation of an anti-tumor drug.

[0009] In the above technical solution, further, the anti-tumor drug is an anti-renal cancer drug.

[0010] In the above technical solution, further, the use is that the enzyme-responsive in situ self-assembled peptide PSK is combined with PD-L1 on the surface of cancer cells, mitochondria are destroyed, and tumor cell apoptosis is induced.

[0011] In the technical solution, further, the application is that the enzyme response in situ self-assembly peptide PSK induces cell immunogenic death.

[0012] In the technical solution, further, the application is to reduce the level of PD-L1, enhance anti-tumor immunity, and induce tumor cell apoptosis.

[0013] In the technical solution, further, the enzyme response self-assembly peptide PSK can form a beta-folded nanofilament structure after being incubated with Legumain.

[0014] In the technical solution, further, the enzyme response self-assembly peptide PSK can escape from lysosomes after entering cells, and PSK and mitochondria show significant co-localization, indicating that PSK can bind to mitochondria after entering cells.

[0015] The application provides an application of a pharmaceutical composition in preparation of an anti-tumor drug, and the pharmaceutical composition comprises the aforementioned enzyme response in situ self-assembly peptide.

[0016] In the technical solution, further, the anti-tumor drug is an anti-renal cancer drug.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] The application designs and synthesizes an in situ self-assembly peptide PSK which targets tumor cells and acts on mitochondria. First, the PD-L1 blocking peptide binds to PD-L1 on the surface of tumor cells, and through endocytosis, the PSK peptide is effectively internalized into tumor cells. In the lysosome, Legumain responds to the bond hydrolysis, triggers self-assembly module assembly to form a beta-folded nanofilament structure, further causes the lysosome membrane permeability to change, escapes from the lysosome, releases KLA to destroy mitochondria, and induces tumor cell apoptosis. At the same time, the PD-L1 blocking peptide is endocytosed into the lysosome, can relieve immunosuppression, and combines with the ICD induced by mitochondrial damage, which will enhance the killing effect of immune cells on tumor cells in the tumor microenvironment. Based on the receptor specificity binding, enzyme response, and self-assembly induced polypeptide accumulation in tumor cells, the selectivity and anti-tumor efficacy of the polypeptide will be effectively improved, and the application is expected to provide a new idea for precise diagnosis and treatment of tumors.

[0019] The self-assembly peptide PSK of the application has good biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The figure is a structural schematic diagram of the PSK of the application;

[0021] Figure 2Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0022] Figure 3 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0023] Figure 4 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0024] Figure 5 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0025] Figure 6 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0026] Figure 7 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0027] Figure 8 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0028] Figure 9 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0029] Figure 10 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0030] Figure 11 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0031] Figure 12 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0032] Figure 13 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0033] Figure 14 Figure A is a schematic diagram of reverse high performance liquid chromatography analysis of the PBP of the present application, Figure B is a schematic diagram of mass spectrometry analysis of the PBP, Figure C is a schematic diagram of reverse high performance liquid chromatography analysis of KLA, Figure D is a schematic diagram of mass spectrometry analysis of KLA, Figure E is a schematic diagram of reverse high performance liquid chromatography analysis of PSK, and Figure F is a schematic diagram of mass spectrometry analysis of PSK;

[0034] Figure 15The diagram illustrates the in vivo antitumor effect of PSK according to the present invention; A. In vivo imaging, B. In vitro imaging, C. Results of HCT 116 tumor cell changes, D. Results of 786-O tumor cell changes, E. TUNEL staining of HCT 116 tumors in each group, F. Changes in body weight of nude mice with HCT 116 tumors in each group, G. TUNEL staining of 786-O tumors in each group, H. TUNEL staining of 786-O tumors in each group. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0036] Example 1: Design, synthesis and characterization of PSK.

[0037] The enzyme-responsive precursor PSK (KLAKLAKKLAKLAK-KLVFF-AAN-CVRARTR) consists of four modules. Figure 1 ), PD-L1 blocking peptide, Legumin enzyme response bond, self-assembly module, mitochondrial toxic peptide KLA.

[0038] The peptide was synthesized using a solid-phase FMOC method, in which amino acids were added sequentially from the carboxyl terminus to the amino terminus. After synthesis, the peptide was cleaved from the resin after removing the side chain protecting groups. In this example, PBP (KLAKLAKKLAKLAK SEQ ID NO.2) and KLA (CVRARTR SEQ ID NO.3) were also synthesized separately during the synthesis of PSK for subsequent comparative experiments. The results showed ( Figure 2 In this embodiment, PBP, KLA, and PSK were successfully synthesized. After purification by high performance liquid chromatography and identification by mass spectrometry, the molecular weights were correct: PBP 861.0 Da, KLA 1524.0 Da, and PSK 3258.5 Da.

[0039] The morphology of the enzyme precursor PSK before and after the addition of Legumain (asparagine endopeptidase) was examined using transmission electron microscopy (TEM). The results showed that... Figure 3 PSK does not aggregate in a sodium acetate buffer solution at pH 5.0, which simulates a lysosomal environment. After incubation with Legumain at 37°C for 2 hours at a final concentration of 100 ng / μl, it transforms into a unique nanofilament structure.

[0040] The secondary structure of PSK was analyzed by circular dichroism spectroscopy. The results showed that ( Figure 4), PSK has a negative peak at 200 nm, which indicates that there is an alpha-helix conformation in PSK. However, after the addition of Legumain, the negative peak at 200 nm disappears, and a negative peak appears around 220 nm, which indicates that the alpha-helix disappears and the beta-sheet structure appears under the action of Legumain. This indicates that after the response of Legumain enzyme, PSK can assemble to form a nanostructure with a beta-sheet conformation.

[0041] Example 2 Cell uptake of PSK

[0042] In order to evaluate the uptake of PSK and KLA in vitro, cell uptake experiments were performed.

[0043] Cy3 dye that can emit red fluorescence was used to label PSK (10 μM) and KLA (10 μM). After incubation in 769-P cells for 5 hours, the fluorescence images were observed, respectively. The results show that Figure 5 ), in the KLA treatment group, no red fluorescence appeared in the 769-P cells. This indicates that the 769-P cells have poor uptake ability for KLA. In the PSK treatment group, the 769-P cells emit red fluorescence, which indicates that the 769-P cells have good uptake ability for PSK.

[0044] In order to more intuitively observe the binding of PSK to mitochondria after entering the cells, a mitochondria co-localization experiment was performed. After 5 hours of treatment with PSK-Cy3, Lyso-Tracker Green working solution and nuclear staining working solution were used to stain 769-P. The results show that Figure 6 ), after PSK treatment, obvious co-localization phenomenon appeared, indicating that after entering the cells, PSK can accurately bind to mitochondria.

[0045] Example 3 PSK effectively kills renal cancer cells with high expression of PD-L1.

[0046] Self-assembling peptide PSK is assembled by PD-L1 blocking peptide PBP and mitochondrial toxic peptide KLA through a series of enzyme response structures and assembly modules. In order to prove that PSK has high safety and more superior tumor cell killing ability, PBP, KLA, equimolar PBP+KLA and PSK were used to treat 293T cells and 769-P cells for 24 hours, respectively, and then the cell viability was compared. The results show that Figure 7), PBP, KLA and PBP+KLA treatment, the killing effect on 769-P cells and 293T cells is very limited, while PSK has obvious killing effect on 769-P cells at low concentration (12.5 μM) and shows dose-dependent. In normal kidney cells 293T, PSK does not affect the viability of 293T cells at the same concentration (12.5 μM), showing better safety.

[0047] Subsequent experiments all use IC50(10 μM) concentration of PSK to treat cells, and the control group uses the same volume of PBS solvent. The treatment condition is to add drug treatment for 24 hours before related detection.

[0048] Next, the expression level of PD-L1 in 293T and 769-P cells was detected by Western-Blot method Figure 8 ), the expression of PD-L1 in 769-P cells is much higher than that in 293T cells. It is inferred that high expression of PD-L1 may mediate the endocytosis of PSK in 769-P, so that PSK can fully exert its anti-tumor effect.

[0049] The expression of PD-L1 after KLA and PSK treatment of 769-P cells was also detected. The results show Figure 9 ), in the KLA treatment group of 769-P cells, the expression of PD-L1 has no obvious difference compared with the control group, while the expression of PD-L1 in the PSK treatment group is significantly down-regulated.

[0050] Example 4 Comparison of the efficacy of KLA and PSK.

[0051] In order to clearly show the influence of PSK on the proliferation ability of cells, 769-P cells in good growth state and in logarithmic growth phase were inoculated in 12-well plates according to the standard of 1000 cells per well. After the cells were completely adherent, 10 μM PSK, 10 μM KLA and PBS were added respectively in DMEM culture medium, and the plates were incubated in the incubator for 7-14 days to analyze the influence on cell clone formation.

[0052] The results show Figure 10 ), there is no significant difference between the KLA treatment group and the control group, and it does not have obvious influence on the proliferation ability of 769-P cells. While in the PSK treatment group, the proliferation ability of 769-P cells is significantly inhibited, indicating that the inhibitory effect of PSK is much stronger than that of KLA.

[0053] In order to further simulate the real tumor growth process, and further prove the permeability of PSK to tumor tissue and the anti-tumor effect, the high-pressure agar medium was evenly covered on the bottom of the 6-well plate, the 769-P cells in the logarithmic growth phase were resuspended, and 1000 cells per well were inoculated in the 6-well plate. The DMEM complete culture medium containing 10 μM PSK and 10 μM KLA was prepared, and PBS was used as a control. After 7-14 days of culture, the spheroid formation of 769-P cells in each treatment group and the control group was observed.

[0054] The results show that Figure 11 ), there was no significant difference between the KLA treatment group and the control group. Compared with the control group and the KLA treatment group, the number of tumor spheres in the PSK treatment group was relatively small, and the volume was also relatively small, indicating that PSK had stronger penetration and killing ability.

[0055] The above results prove that under the same concentration conditions, the inhibition and killing ability of PSK on tumor cell growth is significantly stronger than that of KLA.

[0056] In order to further verify whether PSK can induce cancer cell apoptosis and cause tumor cell death by destroying mitochondria, first, the mitochondrial membrane potential was detected using JC-1 fluorescent probe. 769-P cells were inoculated in a 48-well plate, and after the cells were fully adherent, 10 μM PSK was added for treatment, and a control group (PBS) was reserved, and incubated in an incubator for 24 h. JC-1 has two forms of monomer and polymer. In normal cells, the mitochondrial membrane potential is high, and JC-1 exists in the form of polymer in the mitochondrial matrix, producing red fluorescence; in the early stage of apoptosis, the mitochondrial membrane potential decreases, and JC-1 exists in the form of monomer in the mitochondrial matrix, producing green fluorescence.

[0057] The results show that Figure 12 ), compared with the control group, the red fluorescence was significantly reduced, and the green fluorescence was significantly increased after 24 hours of PSK treatment, indicating that cell apoptosis occurred.

[0058] Induction of ICD is a method of activating tumor cell immunogenicity to enhance anti-tumor immune response, in the process, tumor cells express and release DAMPs, promote T cell infiltration, and convert immune cold tumors into hot tumors. Mitochondrial oxidative stress can trigger a large number of immunogenic death of tumor cells. In view of the fact that destruction of mitochondria can trigger tumor ICD, the induction of ICD by PSK was detected. 769-P cells were inoculated in a 48-well plate, and after the cells were fully adherent, 10 μM PSK, 10 μM KLA were added respectively, and a control group (PBS) was reserved, and incubated in an incubator for 24 h. After the cells were fixed, permeated and blocked, CRT primary antibody and secondary antibody were incubated, and IF staining was performed on the cells in each group to detect the CRT protein egress, so as to detect whether ICD occurred. The results show that the green fluorescence intensity of the CRT in the KLA treatment group and the control group is weak. After PSK treatment, the fluorescence intensity is obviously enhanced, which indicates that the cells in the PSK treatment group have undergone CRT protein egress, thereby proving that PSK induces the occurrence of ICD. Figure 13

[0059] The expression level of Hsp70 protein was also detected. The results show that after 10 μM KLA treatment for 24 hours, the expression level of Hsp70 protein in 769-P cells has no obvious difference with the control group. However, after 10 μM PSK treatment for 24 hours, the expression level of Hsp70 is obviously up-regulated. This result further proves that PSK induces the occurrence of ICD. Figure 14

[0060] Example 5: Anti-tumor effect of PSK in vivo.

[0061] In order to study the bio-distribution of PSK and KLA in vivo, cy5-labeled PSK and KLA were used, and 100 μl of equimolar (200 μM) PSK and KLA were injected into the tail vein of nude mice respectively, and the drug distribution was observed at 6 h and 24 h respectively. Due to the weak tumorigenicity of 769-P cells, multiple attempts failed to construct an effective subcutaneous tumor model, so 786-O cells, which are also kidney cancer cells and are highly sensitive to PSK, were subcutaneously inoculated, and imaging experiments were performed after tumor formation.

[0062] As shown in Figure 15 A, at the 6 h time point, PSK apparently accumulated in the tumor site, while in the KLA treatment group, the drug did not concentrate in the tumor site. The ex vivo imaging results show that Figure 15 B, 24 h after injection of the drug, KLA still did not accumulate in the tumor site, while PSK still apparently accumulated. The above results show that PSK has more obvious tumor targeting ability in vivo.

[0063] ​​To evaluate the antitumor effect of PSK in vivo, tumor models were established by subcutaneously implanting 786-O cells and HCT 116 cells into nude mice. The mice were randomly divided into three groups and injected via tail vein with saline, KLA (2.4 mg / kg), or PSK (5 mg / kg). Results showed that administration of PSK on the third day after inoculation resulted in tumor growth at various time points. Figure 15 Compared with the control group (C(HCT 116), D(786-O)), tumor growth in the KLA treatment group was basically uninhibited, while tumor growth in the PSK treatment group was significantly inhibited. TUNEL staining ( Figure 15 E(HCT116 cells), Figure 15 G(786-O cells) showed that PSK significantly killed tumors. Body weight changes in mice of each group were recorded during drug administration, and the data showed ( Figure 15 The body weight of nude mice in groups F and H did not change significantly, indicating that PSK has no obvious toxic effects. These results demonstrate that PSK has high biocompatibility in vivo and causes almost no damage to normal tissues and organs.

Claims

1. An enzyme-responsive in-situ self-assembled peptide PSK under the action of tumor microenvironment enzymes, characterized in that, The amino acid sequence of the self-assembled peptide PSK is KLAKLAKKLAKLAKKLVFFAANCVRARTR.

2. Use of the enzyme-responsive in-situ self-assembled peptide PSK in the preparation of a drug for resisting kidney cancer according to claim 1.

3. Use according to claim 2, characterized in that, The application is that the enzyme-responsive in-situ self-assembled peptide PSK binds to the PD-L1 on the surface of the cancer cell, destroys the mitochondria, and induces the apoptosis of the tumor cell.

4. Use according to claim 2, characterized in that, The application is that the enzyme-responsive in-situ self-assembled peptide PSK induces the occurrence of the immunogenic death of the cell.

5. Use according to claim 2, characterized in that, The application is to reduce the level of PD-L1, enhance the anti-tumor immunity, and induce the apoptosis of the tumor cell.

6. Use of a pharmaceutical composition in the preparation of a drug for resisting kidney cancer, wherein the pharmaceutical composition comprises the enzyme-responsive in-situ self-assembled peptide PSK according to claim 1.

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