Polypeptide for inducing immunogenic death of tumor cells, bioactive solution and application thereof

Abstract

The invention provides a polypeptide for inducing tumor cell immunogenic death, a bioactive solution and application of the polypeptide and the bioactive solution, and belongs to the technical field of biological medicine. Comprising an end-capping group, a phosphorylated gelling factor, a matrix metalloproteinase-2 response sequence and a transient receptor potential vanillic acid receptor 1 (TRPV1) agonist which are connected in sequence, the amino acid sequence of the phosphorylated gelling factor is GFFpY, and the response sequence of the matrix metalloproteinase-2 is PLGVR. The anti-tumor polypeptide provided by the invention can generate enzymatic in-situ self-assembly through the action of ALP and MMP-2 overexpressed outside tumor cells, and sequentially release pharmacophores outside and inside the cells to cause mitochondrial stress and endoplasmic reticulum stress and synergistically induce immunogenic death of the tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a polypeptide for inducing immunogenic death of tumor cells, a bioactive solution and applications thereof. Background Art

[0002] Cancer immunotherapy, especially adoptive cell transfer therapy and immune checkpoint blockade, has provided a thriving impetus for cancer treatment by harnessing the immune system to treat malignancies. However, tumor heterogeneity, including differences in tumor antigen and immune checkpoint expression, limits their clinical application, and these challenges highlight the urgent need for innovation and progress in cancer immunotherapy.

[0003] Immunogenic cell death (ICD) has been shown to transform immune “cold” tumors into “hot” tumors, showing good promise in reshaping the immunosuppressive microenvironment and improving tumor immunogenicity by inducing the release of damage-associated molecular patterns (DAMPs), such as calreticulin (CRT), high-mobility group box 1 (HMGB1), and adenosine 5'-triphosphate (ATP). Although some chemotherapeutic drugs have ICD-inducing functions, such as doxorubicin, oxaliplatin, and paclitaxel, and can be used in combination with immune checkpoint blockers (ICBs), their efficacy is limited. In addition, most other common chemotherapeutic drugs do not have satisfactory ICD-inducing efficacy due to extremely poor solubility, low circulating pharmacokinetics, and insufficient tumor accumulation and retention. In view of these limitations, alternative strategies are being explored to improve the ICD-inducing effect of chemotherapeutic drugs. Combinations of different chemotherapeutic drugs, even if they cannot effectively induce ICD when present alone, may work synergistically to improve their signaling and induce sustained antitumor immune responses. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a polypeptide for inducing immunogenic death of tumor cells, a bioactive solution and applications thereof; the polypeptide for inducing immunogenic death of tumor cells provided by the present invention can undergo enzymatic in situ self-assembly through the action of alkaline phosphatase (ALP) and matrix metalloproteinase-2 (MMP-2) overexpressed outside tumor cells, sequentially release pharmacophores outside and inside cells, cause mitochondrial stress and endoplasmic reticulum stress, and synergistically induce immunogenic death of tumor cells.

[0005] A polypeptide inducing immunogenic death of tumor cells comprises a capping group, a phosphorylated gelling factor, a matrix metalloproteinase-2 response sequence and a transient receptor potential vanilloid receptor 1 (TRPV1) agonist connected in sequence; the amino acid sequence of the phosphorylated gelling factor is GFFpY, and the matrix metalloproteinase-2 response sequence is PLGVR.

[0006] Preferably, the capping group is camptothecin (CPT), and the TRPV1 agonist is synthetic capsaicin (Caps).

[0007] Further preferably, the anti-tumor polypeptide has a structural formula as shown in Formula I:

[0008] Formula I.

[0009] The invention provides a bioactive solution, comprising the polypeptide inducing immunogenic death of tumor cells.

[0010] Preferably, the bioactive solution uses phosphate buffered saline (PBS) as a solvent.

[0011] Preferably, the pH of the PBS is 7.2-7.4.

[0012] Preferably, the mass volume ratio of the anti-tumor polypeptide to the buffer is 4 mg: 0.8~1.2 mL.

[0013] The present invention provides the use of the polypeptide inducing immunogenic death of tumor cells and the bioactive solution in the preparation of anti-tumor immune drugs.

[0014] Preferably, the anti-tumor immune drug includes an anti-breast cancer drug.

[0015] Compared with the prior art, the present invention has the following beneficial effects: the polypeptide inducing immunogenic death of tumor cells provided by the present invention can be dephosphorylated and cleaved under the action of ALP and MMP-2 overexpressed outside tumor cells, releasing the active derivative VRK-Caps of capsaicin outside the cells, and self-assembling in situ to form nanofibers; after the nanofibers are taken up by cells, CPT is slowly released under the action of intracellular proteases. VRK-Caps activates TRPV1 channels, leading to extracellular calcium influx, causing cellular calcium overload and mitochondrial stress; CPT induces endoplasmic reticulum stress in cells. The synergistic effect of the two effectively causes immunogenic death of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The high-resolution mass spectrum and liquid chromatography at an absorption wavelength of 254 nm of the polypeptide CPMC synthesized in Example 3 that induces immunogenic death of tumor cells;

[0017] Figure 2 The high-resolution mass spectrum and liquid chromatography at an absorption wavelength of 254 nm of the polypeptide CPM synthesized in Comparative Example 1;

[0018] Figure 3The high-resolution mass spectrum and liquid chromatography at an absorption wavelength of 254 nm of the polypeptide CMC synthesized in Comparative Example 2;

[0019] Figure 4 The high-resolution mass spectrum and liquid chromatography at an absorption wavelength of 254 nm of the polypeptide CPC synthesized in Comparative Example 3;

[0020] Figure 5 The high-resolution mass spectrum and liquid chromatography at an absorption wavelength of 254 nm of the polypeptide NPMC synthesized in Comparative Example 4;

[0021] Figure 6 is the chemical structural formula of the polypeptide synthesized in Comparative Examples 1-4;

[0022] Figure 7 The liquid chromatogram of CPMC before and after co-catalysis by ALP and MMP-2 in Experimental Example 1;

[0023] Figure 8 The microscopic morphology of the PBS solution of CPMC in Experimental Example 1 before and after co-catalysis by ALP and MMP-2;

[0024] Fig. 9 The confocal image (top) and the mean fluorescence intensity (bottom) of the intracellular calcium ion content detected in Experimental Example 2;

[0025] Fig.10 It is a confocal image of mitochondrial membrane potential in Experimental Example 3;

[0026] Fig.11 It is the Western Blot band diagram of the endoplasmic reticulum stress marker protein in Experimental Example 4;

[0027] Fig.12 The confocal image (upper) and flow cytometry image (lower) of CRT eversion in Experimental Example 5;

[0028] Fig.13 This is a statistical diagram of tumor volume in Experimental Example 6;

[0029] Fig.14 This is an optical photograph of the tumor after ex vivo treatment in Experimental Example 6. DETAILED DESCRIPTION

[0030] The present invention provides a polypeptide for inducing immunogenic death of tumor cells, comprising a capping group, a phosphorylated gelling factor, a matrix metalloproteinase-2 response sequence and a transient receptor potential vanilloid receptor 1 (TRPV1) agonist connected in sequence; the amino acid sequence of the phosphorylated gelling factor is GFFpY, and the matrix metalloproteinase-2 response sequence is PLGVR.

[0031] In the present invention, the capping group is preferably camptothecin (CPT); the TRPV1 agonist is synthetic capsaicin (Caps); the anti-tumor polypeptide is composed of an amino acid sequence of L configuration; an alkaline phosphatase reaction site exists in the tyrosine in the anti-tumor polypeptide; further preferably, the structural formula of the polypeptide that induces immunogenic death of tumor cells is as shown in Formula I:

[0032] Formula I.

[0033] The present invention has no particular limitation on the method for preparing the polypeptide that induces immunogenic death of tumor cells, and any conventional preparation method in the art may be used. In the specific implementation of the present invention, the Fmoc-short peptide solid phase synthesis method is preferably used.

[0034] The present invention provides a bioactive solution, comprising the polypeptide that induces immunogenic death of tumor cells. Preferably, the bioactive solution uses PBS buffer as a solvent; the pH of the PBS buffer is preferably 7.2-7.4, more preferably 7.3; in the present invention, the mass volume ratio of the anti-tumor polypeptide to the buffer is preferably 4 mg: 0.8-1.2 mL, more preferably 0.4 mg: 0.9-1.1 mL. In the present invention, the bioactive solution is preferably obtained by mixing the polypeptide that induces immunogenic death of tumor cells with the PBS buffer.

[0035] The present invention provides the use of the polypeptide inducing immunogenic death of tumor cells and the bioactive solution in the preparation of anti-tumor immune drugs. In the present invention, the dosage form of the anti-tumor immune drug is preferably an injection preparation; the anti-tumor immune drug preferably includes an anti-breast cancer drug.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] Example 1

[0039] Synthesis of CPT-NPC

[0040] Camptothecin (CPT, 0.437 g, 1.25 mmol) and dimethylaminopyridine (DMAP, 0.91 g, 7.5 mmol) were dissolved in dichloromethane (DCM, 10 ml) and stirred under ice bath. After 20 minutes, chloromethyl 4-nitrobenzoate (p-NPC, 0.5 g, 2.5 mmol) in DCM (10 mL) was slowly added to the reaction system, and then stirred at 40 °C for 4 hours.

[0041] The mixture was washed three times with 1N hydrochloric acid solution, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure by rotary evaporation. Ether was added for recrystallization, and a yellow solid precipitated. The yellow solid product was filtered and dried to obtain CPT-NPC (482 mg, 75%), which could be used for the subsequent reaction.

[0042] Example 2

[0043] Synthesis of Caps-NPC

[0044] Synthetic capsaicin (Caps, 0.73 g, 2.5 mmol), p-NPC (1.07 g, 5 mmol) and DIEA (1.65 mL, 10 mmol) were dissolved in DCM (10 mL) and stirred at 25 ° C for 4 hours. The reaction mixture was then washed three times with 1N aqueous hydrochloric acid, dried over anhydrous sodium sulfate, filtered, and then concentrated to a viscous liquid by rotary evaporation under reduced pressure. After adding diethyl ether, Caps-NPC precipitated as a white solid, which was filtered and dried (0.5 g, 43.6%) and used for the subsequent reaction.

[0045] Example 3

[0046] Synthesis of peptide CPT-GFFpY-PLGVRK-Caps

[0047] The peptide obtained by combining the classic Fmoc-short peptide solid phase synthesis method with the liquid phase synthesis method is referred to as CPMC. The specific steps are:

[0048] 1) Weigh 0.5 mmol 2-Cl-Trt resin into a solid phase synthesis tube, add 15 mL DCM, and shake on a shaker for 10 minutes to allow the 2-Cl-Trt resin to fully swell;

[0049] 2) Use an ear bulb to completely squeeze out DCM from the solid phase synthesizer containing 2-Cl-Trt resin;

[0050] 3) Dissolve 0.75 mmol of Fmoc-protected lysine (Fmoc-Lys(Boc)-OH) in 10 mL of DCM, add 1.5 mmol of N,N-diisopropylethylamine (DIEA), mix well, add to the above solid phase synthesizer, and place on a shaker to react at room temperature for 1 hour;

[0051] 4) Blocking: Use an ear bulb to remove the reaction solution in the solid phase synthesis tube, then wash with 10 mL DCM each time, 1 minute each time, for a total of 3 times, add 16 mL of a solution with a volume ratio of DCM: DIEPA: methanol of 17:1:2, and react at room temperature for 15 minutes;

[0052] 5) Use an ear bulb to remove the reaction solution in the solid phase synthesis tube, first wash with DCM, use 10 mL of DCM each time, wash for 1 minute, wash 3 times in total, then wash with N,N-dimethylformamide (DMF), use 10 mL of DMF each time, wash for 1 minute, wash 3 times in total, add 15 mL of DMF containing 20% ​​piperidine (volume ratio), react for 30 minutes, then wash with DMF, use 10 mL of DMF each time, wash for 1 minute, wash 5 times in total;

[0053] 6) Weigh 1 mmol of the second Fmoc-protected arginine (Fmoc-Arg(Pbf)-OH), 1 mmol of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and 2 mmol of DIEA, dissolve them in 10 mL of DMF, add the dissolved amino acid solution to the above solid phase synthesis tube, and react for 1 hour;

[0054] 7) Repeat steps 5) and 6) to add amino acids sequentially until the last amino acid at the C-terminus;

[0055] 8) Dissolve 1 mmol of CPT-NPC and 2 mmol of DIEA in DCM, add to the solid phase synthesis tube, react overnight, wash with DMF 5 times, wash with dichloromethane 5 times, and proceed to the next step;

[0056] 9) Prepare 10 mL of cutting solution, which is 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% H2O (volume ratio), add it to the above solid phase synthesizer, react at room temperature for 1 hour, cut the product from the 2-Cl-Trt resin, and remove the solvent with a rotary evaporator to obtain a crude product;

[0057] 10) Caps-NPC (240 mg, 0.52 mmol), CPT-GFFpYPLGVRK (854 mg, 0.52 mmol) and DIEA (600 μL, 3.62 mmol) were dissolved in 12 mL DMF and stirred at room temperature overnight;

[0058] 11) Then, semi-preparative high performance liquid chromatography was used for separation and purification to obtain the polypeptide of Example 3. The polypeptide obtained in Example 3 was detected by high resolution mass spectrometry and high performance liquid chromatography. The results are shown in Figure 1 The structural formula of the polypeptide of Example 3 is shown in Formula I.

[0059] Comparative Example 1

[0060] Synthesis of peptide CPT-GFFpY-PLGVRK

[0061] The polypeptide of Comparative Example 1 was prepared according to the Fmoc-short peptide solid phase synthesis method of Example 1. The obtained polypeptide is referred to as CPM. When synthesizing the polypeptide of Comparative Example 1, Caps is missing compared with the method of Example 3. The polypeptide obtained in Comparative Example 1 was detected by high-resolution mass spectrometry and high-performance liquid chromatography. The results are shown in Figure 2 , see the structural formula Figure 6 .

[0062] Comparative Example 2

[0063] Synthesis of peptide CPT-GFFY-PLGVRK-Caps

[0064] The polypeptide of Comparative Example 2 was prepared according to the synthesis method of Example 1. The obtained polypeptide is referred to as CMC. When synthesizing the polypeptide of Comparative Example 2, the tyrosine lacks a phosphate group compared with the method of Example 3. The polypeptide obtained in Comparative Example 2 was tested by high-resolution mass spectrometry and high-performance liquid chromatography. The results are shown in FIG. Figure 3 , see the structural formula Figure 6 .

[0065] Comparative Example 3

[0066] Synthesis of peptide CPT-GFFpY-PRLVGK-Caps

[0067] The polypeptide of comparative example 2 was prepared according to the synthesis method of example 1, and the obtained polypeptide is referred to as CPC. When synthesizing the polypeptide of comparative example 3, the MMP-2 response sequence was disrupted compared with the method of example 3. The polypeptide obtained in comparative example 3 was detected by high-resolution mass spectrometry and high-performance liquid chromatography, and the results are shown in Figure 4 , see the structural formula Figure 6 .

[0068] Comparative Example 4

[0069] Synthesis of peptide Nap-GFFpY-PRLVGK-Caps

[0070] The polypeptide of Comparative Example 4 was prepared according to the solid phase synthesis method of Example 1. The obtained polypeptide is referred to as NPMC. When synthesizing the polypeptide of Comparative Example 4, the end capping group CPT was replaced with β-naphthylacetic acid (Nap) compared with the method of Example 3. The polypeptide obtained in Comparative Example 4 was detected by high resolution mass spectrometry and high performance liquid chromatography. The results are shown in Figure 5 , see the structural formula Figure 6 .

[0071] Experimental Example 1

[0072] Enzyme-catalyzed transformation and microscopic morphology characterization

[0073] Weigh 3.92 mg of the polypeptide prepared in Example 3, add 1 mL of 1×PBS (pH=7.4) solution, add 1 M sodium carbonate solution in small amounts and multiple times to adjust the pH value until it is completely dissolved (final pH=7.4), then add ALP (10 U mL -1 ) and MMP-2 (5 μg mL -1 ), placed in a 37°C thermostat and reacted for 12 hours. Before and after enzyme catalysis, 10 μL of the mother solution was diluted with 90 μL of methanol, and the conversion was detected by high performance liquid chromatography. The results are shown in Figure 7 In addition, transmission electron microscope samples were prepared before and after adding enzyme to observe their microscopic morphology. Figure 8 .

[0074] Figure 7 and Figure 8 It shows that the peptide CPMC for inducing immunogenic death of tumor cells prepared in Example 3 can respond well to ALP and MMP-2, and forms two parts, VRK-Caps and CPT-GFFY-PLG, after catalysis. There is no obvious assembly structure before adding enzymes, and after adding ALP and MMP-2, CPMC self-assembles into nanofibers.

[0075] Experimental Example 2

[0076] Intracellular calcium ion content detection

[0077] Eight groups were set up, namely PBS (Control), CPT + Caps, CPM, CMC, CPC, NPMC, CPMC, CPMC + Cpz (Cpz is Capsazepin, a TRPV1 specific inhibitor). 5Mouse breast cancer 4T1 cells were plated on confocal dishes at a density of 100 μM. After the cells adhered overnight, 1 mL of culture medium containing 100 μM of the above substances was replaced and incubated for 8 hours (the CPMC + Cpz group was pre-treated with 1 μM Cpz for 30 minutes). Wash with HBSS, then dilute 2 mM Fluo-4AM to 1 μM with HBSS, add 0.02% Pluronic F-127, and incubate with the cells at 37°C in the dark for 20 minutes. Then wash with Hank's balanced salt solution (HBSS), stain the nucleus with 1× Hochest 33342 dye diluted in PBS for 10 minutes, and then incubate at 37°C for another 10 minutes. Images were taken at the same voltage using a laser scanning confocal microscope (Leica TSC SP8) to detect changes in intracellular calcium ion concentration after different treatments, such as Fig. 9 As shown in the figure above. The green fluorescence was brightest in the NPMC and CPMC groups, followed by CPC; the fluorescence of CPMC weakened after the use of TRPV1 inhibitor. There was no obvious fluorescence in the other groups. This indicates that after MMP-2 cleaves and releases active VRK-Caps, it activates TRPV1 and causes extracellular calcium ion influx.

[0078] The calcium concentration after the same treatment was semi-quantitatively analyzed by flow cytometry. 5 Mouse breast cancer 4T1 cells were plated in a 12-well plate at a density of / well. After the cells adhered overnight, 1 mL of culture medium containing 100 μM of the above-mentioned different substances was replaced and incubated for another 8 hours (the CPMC + Cpz group was pre-treated with Cpz for 30 minutes). The cells were digested with trypsin, transferred to a flow tube, and centrifuged at 1000 g for 3 minutes. 2 mM Fluo-4 AM was diluted to 1 μM with HBSS, 0.02% Pluronic F-127 was added, and incubated with the cells at 37°C in the dark for 20 minutes. After washing with HBSS, the cells were placed in an incubator for an additional 20 minutes before being tested on the machine. The statistical results of the mean fluorescence intensity MFI of the calcium ion probe were consistent with the confocal trend, as shown in the following figure. Fig. 9 As shown in the figure below.

[0079] Experimental Example 3

[0080] Mitochondrial membrane potential measurement

[0081] The experimental groups, confocal dishes and drug incubation were set up according to the method of Experimental Example 2, and the drug incubation time for each group was 4 hours. After washing with PBS, 200 nM Mito-Tracker Red CMXRos was used to incubate the cells with the cells at 37 °C in the dark for 30 minutes. After the incubation, the staining solution was removed and 500 μL of serum-free culture medium was added. Images were taken at the same voltage using a laser scanning confocal microscope (Leica TSC SP8) to detect the changes in mitochondrial membrane potential in cells after different treatments, such as Fig.10 As shown in the figure, the red fluorescence of the probe almost disappeared after incubation with CPMC and NPMC, which means that the mitochondrial membrane potential was severely reduced, causing mitochondrial stress; the presence of Cpz restored the situation in the CPMC group. This is consistent with the changes in calcium ion concentration.

[0082] Experimental Example 4

[0083] Detection of endoplasmic reticulum stress-related protein expression

[0084] Seven groups were set up, namely PBS (Control), CPT + Caps, CPM, CMC, CPC, NPMC, and CPMC. 5 Mouse breast cancer 4T1 cells were plated in 6-well plates at a density of 1 / well. After the cells adhered overnight, 2 mL of culture medium containing 100 μM of the above substances was replaced and incubated for 24 hours. The whole cell protein was then extracted with RIPA (strong) lysis buffer, and protein expression was analyzed by standard Western Blot operation, such as Fig.11 As shown. It can be seen that the increase in the expression of the three endoplasmic reticulum stress-related proteins is mainly due to the effect of CPT. CPT + Caps, CPM, CPC, and CPMC all induced similar levels of upregulation of protein kinase R-like endoplasmic reticulum kinase (PERK), transcription activator 6 (ATF6), and phosphorylated α-subunit eukaryotic initiation factor 2 (p-EIF2α). CMC had no obvious effect due to solubility issues, while NPMC did not contain CPT groups.

[0085] Experimental Example 5

[0086] Detection of calreticulin externalization

[0087] Seven groups were set up, namely PBS (Control), CPT + Caps, CPM, CMC, CPC, NPMC, and CPMC. 5The mouse breast cancer 4T1 cells were spread on a confocal dish at a density of 100 μM. After the cells adhered overnight, 1 mL of culture medium containing 100 μM of the above-mentioned different substances was replaced and incubated for 24 hours. After the incubation, the cells were fixed and blocked, and incubated with the CRT primary antibody in a 4°C refrigerator for 12 hours. Then, the cells were taken out of the refrigerator to return to room temperature and incubated with the fluorescently labeled secondary antibody for 1 hour. After washing, the nuclei were stained with DAPI for 10 minutes. Images were taken at the same voltage using a laser scanning confocal microscope (Leica TSC SP8) to detect the externalization of CRT to the cell membrane after different treatments, such as Fig.12 As shown in the figure above, cells treated with CPMC showed the brightest red fluorescence, followed by CPC, and almost no red fluorescence was visible in the other groups, indicating that CPMC induced the most CRT eversion.

[0088] CRT externalization was semi-quantitatively analyzed by flow cytometry after the same treatment. 5 Mouse breast cancer 4T1 cells were plated in a 12-well plate at a density of 1 / well. After the cells adhered overnight, 1 mL of culture medium containing 100 μM of the above substances was replaced and incubated for 24 hours. The cells were digested with trypsin, transferred to a flow tube, and centrifuged at 1000 g for 3 minutes. After washing with PBS, the cells were incubated on ice with a fluorescently labeled CRT primary antibody for 30 minutes, and then tested on the machine. The results are shown in Figure 2. Fig.12 As shown in the figure below, the proportion of CRT+ cells in the CPMC group increased to 60.3%, followed by 20.2% in the CPC group. Compared with the control group with only CPT group or Caps group or the combination of two free drugs, the effect was significantly improved.

[0089] CRT eversion is the gold standard for determining the occurrence of ICD. In Example 3 and Comparative Examples 1-4, only when endoplasmic reticulum stress and mitochondrial stress caused by calcium overload coexisted did obvious ICD occur. This confirms that our dual enzyme response system is crucial to the effect of CPT and Caps synergistically inducing ICD, and CPMC has excellent ICD induction potential.

[0090] Experimental Example 6

[0091] Animal tumor models

[0092] A bilateral subcutaneous tumor-bearing model was established using mouse breast cancer 4T1 cells. The mice used were female Balb / c mice (6-8 weeks, 16 g ~19 g). When the primary tumor of the mouse reached 70 mm in size, 3At 4 pm, the mice were randomly divided into PBS group, CPT + Caps group, CPM group, CMC group, CPC group, NPMC group, CPMC group and CPMC + aPDL1 group (aPDL1, i.e. programmed cell death-ligand 1 monoclonal antibody), with 5 mice in each group. Tumor volume calculation formula: length × width 2 / 2. The drug was administered by intratumoral injection at the proximal tumor with a concentration of 14 µmol / kg on days 1, 3, and 5. For the CPMC + aPDL1 group, aPDL1 (2.5 mg / kg) was injected intraperitoneally on days 2, 4, and 6. The tumor growth trend of each group of mice was monitored. Fig.13 The results showed that CPMC can effectively inhibit the growth of proximal and distal tumors, and the effect is better when used in combination with aPDL1. This shows that after CPMC kills the proximal tumor, it causes a systemic immune response in the body through ICD, thereby inhibiting the growth of the distal tumor. The mice were killed at the end of the experiment on the 15th day. The tumors in each group were taken out and photographed, as shown in the figure below. Fig.14 The results showed that the tumors in mice in the CPMC group and CPMC + aPDL1 were very small, indicating an excellent anti-tumor effect.

[0093] From the above experimental examples, it can be seen that the polypeptides inducing immunogenic death of tumor cells provided by the present invention, through the action of ALP and MMP-2 overexpressed outside tumor cells, undergo enzymatic in situ self-assembly, sequentially release pharmacophores outside and inside cells, cause mitochondrial stress and endoplasmic reticulum stress, and synergistically induce immunogenic death of tumor cells. The in vivo tumor experiment in mice proved that the polypeptides of the present invention can effectively inhibit the growth of proximal tumors and distal tumors.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide that induces immunogenic death of tumor cells, characterized in that: It comprises a capping group, a phosphorylated gelling factor, a matrix metalloproteinase-2 response sequence and a transient receptor potential vanilloid receptor 1 agonist connected in sequence; the amino acid sequence of the phosphorylated gelling factor is GFFpY, and the matrix metalloproteinase-2 response sequence is PLGVR.

2. The polypeptide that induces immunogenic death of tumor cells according to claim 1, characterized in that The blocking group is camptothecin.

3. The polypeptide that induces immunogenic death of tumor cells according to claim 2, characterized in that: The transient receptor potential vanilloid receptor 1 agonist is synthetic capsaicin.

4. The polypeptide that induces immunogenic death of tumor cells according to claim 3, characterized in that The structural formula of the polypeptide that induces immunogenic death of tumor cells is shown in Formula I: Formula I.

5. A biologically active solution, characterized in that A polypeptide that induces immunogenic death of tumor cells according to any one of claims 1 to 4.

6. The bioactive solution according to claim 5, characterized in that The biologically active solution uses phosphate buffer as solvent.

7. The biologically active solution according to claim 6, characterized in that The pH of the phosphate buffer is 7.2-7.

4.

8. The bioactive solution according to any one of claims 5 to 7, characterized in that: The mass volume ratio of the polypeptide inducing immunogenic death of tumor cells to the phosphate buffer is 4 mg: 0.8-1.2 mL.

9. Use of the polypeptide inducing immunogenic death of tumor cells according to any one of claims 1 to 4 and the bioactive solution according to any one of claims 5 to 7 in the preparation of anti-tumor immune drugs.

10. The use according to claim 9, characterized in that: The anti-tumor immune drugs include anti-breast cancer drugs.

Images