Antigen targeting peptide nano-micelle and application thereof

Abstract

The invention discloses an antigen targeting peptide nano-micelle and application thereof, belongs to the field of biological medicine, and particularly relates to an antigen targeting peptide molecule which is composed of polyethylene glycol as a hydrophilic component and a hydrophobic region containing a matrix metalloproteinase-2 restriction site, preS1 antigen peptide and low-pH insertion peptide. The antigen targeting peptide nano-micelle is prepared from antigen targeting peptide molecules through a self-assembly technology. The antigen targeting peptide nano-micelle disclosed by the invention has accurate tumor microenvironment responsiveness, antigen peptide can be released in TME, and a preS1 epitope is anchored to a tumor cell membrane through pHLIP, so that a virus mechanism is simulated. According to the present invention, the tumor immune microenvironment can be remodeled, and the tumor cells can be induced to display the virus antigen peptide so as to activate the pre-existing anti-virus immune reaction, such that the antibody-mediated tumor cell killing can be achieved.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to an antigen targeting peptide nano-micelle and a use thereof. Background Art

[0002] Tumors are still an important factor that poses a serious threat to people's lives and health. Although my country has developed a variety of treatment methods, the number of deaths caused by tumors remains high. Especially for patients with advanced tumors, surgical treatment is often not suitable. Chemotherapy, as an important treatment method, has played a certain role in tumor control, but conventional chemotherapy has low selectivity and usually requires high doses of drugs to kill tumor cells, which often leads to significant toxic side effects. In contrast, immunotherapy has brought new hope for the treatment of solid tumors. Immunotherapy activates the patient's own immune system to identify and attack tumor cells. It has high selectivity and durability and can significantly reduce damage to normal cells. With the continuous development of immunotherapy, more and more patients with solid tumors have achieved better prognosis and greatly improved their quality of life. This emerging treatment method provides a more effective option for patients with advanced tumors and has become an important breakthrough in the field of tumor treatment.

[0003] B cells are a strategy to fight tumors by regulating or enhancing their functions in anti-cancer immunotherapy. As an important part of the immune system, B cells mainly fight tumors by producing antibodies and participating in immune responses. In recent years, a series of advances have been made in anti-cancer immunotherapy research targeting B cells, including monoclonal antibody therapy and tumor vaccine therapy. These therapies have shown good efficacy in tumor types such as lymphoma and breast cancer, and have become an important part of tumor immunotherapy.

[0004] However, optimal target selection remains a key challenge in the development of cancer immunotherapy, which directly affects the treatment effect and patient prognosis. In cancer cells, many potential therapeutic targets are not limited to the tumor cells themselves, but may also be expressed in other cell types in the tumor microenvironment, such as immune cells, vascular endothelial cells, and stromal cells. This significant expression heterogeneity in different tumor regions and multiple cell subpopulations increases the complexity of target selection and may even lead to differences in treatment effects. In addition, dynamic changes during tumor evolution and treatment, such as cell mutations, clonal selection, and changes in the microenvironment, can lead to continuous changes in target expression patterns. Such changes may make it possible for originally effective targets to lose their effect during treatment, making it more difficult to achieve lasting therapeutic effects. Therefore, it is particularly important to develop highly adaptable immunotherapy strategies. At the same time, for some rare tumors or specific subtypes, research on target expression is relatively limited, resulting in a lack of highly targeted immunotherapy regimens in the clinic. This research gap not only limits the formulation of immunotherapy strategies, but also makes doctors lack effective treatment options when facing specific patients.

[0005] Emerging strategies to address the bottleneck of therapeutic targets mainly focus on regulating target expression through methods such as gene editing, small molecule drugs and antibody intervention. However, these methods often face challenges in efficiency, safety and drug resistance, which limit their widespread promotion in clinical applications. In addition, the introduction of therapeutic targets into tumors has opened up new avenues for immunotherapy. Taking advantage of the ability of certain bacteria to selectively colonize in the core of immune-privileged tumors, synthetic biology techniques can be used to specifically release and label antigens recognized by CAR-T cells in tumor tissues. This strategy can not only enhance the targeting and killing effects of CAR-T cells, but also improve the therapeutic effect by inducing local immune responses. However, this approach requires the careful design of CAR-T cells to ensure their effectiveness and safety. For example, CAR-T cells must be able to effectively recognize and attack tumor cells in the tumor microenvironment while avoiding damage to normal cells.

[0006] It is noteworthy that individuals who receive planned immunizations, such as hepatitis B vaccination, have a reserve of antiviral immune memory. These immune memory cells, such as B cells, can quickly activate and produce a large number of neutralizing antibodies when attacked by viruses or in the presence of viral antigens, thereby effectively clearing the infection. This immune memory mechanism provides the body with long-lasting protection, allowing the immune system to respond quickly in a short period of time when exposed to the same virus again. However, the specificity of these immune responses limits the ability of antiviral memory to effectively recognize and attack tumor cells. Tumor cells often have antigens that are different from normal cells, and the expression and variation of these antigens make it difficult for the immune system to recognize them as foreign threats. In addition, the immunosuppressive effects of the tumor microenvironment further weaken the ability of antiviral memory cells to attack tumors. Therefore, converting antiviral immunity into antitumor immunity may become a promising new direction in cancer treatment. The key to this conversion lies in how to adjust and enhance existing immune memory so that it can not only target viral antigens but also effectively recognize tumor-associated antigens. Summary of the invention

[0007] The purpose of the present invention is to provide an antigen targeting peptide nano-micelle and its use, which can improve the immune microenvironment in the tumor, has good tumor targeting ability, activates antiviral immune memory after being released in the tumor microenvironment, can be inserted into the surface of tumor cells, has strong adaptability, avoids damage to normal cells and converts antiviral immunity into anti-tumor immunity.

[0008] The present invention relates to an antigen-targeted nano-micelle, which can effectively release antigens that mark tumor tissues, thereby achieving in situ lysis of tumors and overcoming the challenge of identifying targets with specific and consistent expression in heterogeneous tumors.

[0009] The antigen-targeted nanomicelles provided by the present invention showed significant therapeutic effects in a head and neck squamous cell carcinoma model. The nanomicelles effectively inhibited the growth, recurrence and metastasis of tumors in animals pre-immunized with preS1. The experimental results showed that the enhancement of the therapeutic effect was closely related to the increase in the proportion of preS1-specific B cells and activated tumor-specific T cells in the tumor microenvironment.

[0010] The nano-micelles of the present invention have the function of disguising tumor cells as viruses, and by inducing tumor cells to express viral antigen peptides, simulating "giant viruses", thereby activating the pre-existing antiviral immune response and effectively reshaping the tumor immune microenvironment. In this way, tumor cells can be targeted by pre-existing anti-hepatitis B virus antibodies, thereby activating the antibody-mediated tumor cell killing mechanism and achieving effective tumor killing.

[0011] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: An antigen targeting peptide molecule is as follows: PEG polymer-PLGLAG-preS1-pHLIP, the PEG polymer includes any one of PEG and R-PEG, the molecular weight of the PEG is 1000-3000, the R contains lactic acid fragments and 5-hydroxynicotinic acid fragments, and the molecular weight of the PEG in the R-PEG is 1000-3000. The present invention forms an antigen targeting peptide molecule by a PEG polymer including PEG or R-PEG and a PLGLAG-preS1-pHLIP with a specific structure, which can be used in the treatment of tumors. The antigen targeting peptide molecule synthesized by the present invention has the effects of improving the immune microenvironment in the tumor, having good tumor targeting ability, activating antiviral immune memory after being released in the tumor microenvironment, being able to be inserted into the surface of tumor cells, having strong adaptability, avoiding damage to normal cells, and converting antiviral immunity into antitumor immunity.

[0012] Preferably, the antigen targeting peptide molecule is as follows: PEG2000-PLGLAG-preS1-pHLIP; or, in the preparation of R-PEG, lactic acid and 5-hydroxynicotinic acid are pre-polymerized and then polymerized with PEG1500.

[0013] Preferably, PLGLAG is an MMP2 restriction site, preS1 is an HBV antigen peptide, and pHLIP is a low pH insertion peptide with a sequence of ACDDQNPWRAYDLLFPTDTLLLDLLW.

[0014] The invention discloses an antigen targeting peptide nano-micelle, comprising: the above-mentioned antigen targeting peptide molecule.

[0015] Preferably, the antigen targeting peptide nano-micelles are formed by self-assembly of antigen targeting peptide molecules in phosphate buffered saline.

[0016] Preferably, the pH of phosphate buffered saline is 6.5-7.8; the concentration of the antigen targeting peptide molecules added to phosphate buffered saline is 1.23-30 μM.

[0017] The present invention discloses a method for preparing a cell model with preS1 peptide inserted on its surface, comprising: treating the above-mentioned antigen targeting peptide nano-micelles with MPP2, and then mixing the antigen targeting peptide nano-micelles treated with MPP2 with SCC7 cells for incubation for 1-48 hours to prepare a cell model with preS1 peptide inserted on its surface.

[0018] The invention discloses a method for preparing an antigen targeting peptide molecule, comprising: reacting a PEG polymer with PLGLAG-preS1-pHLIP in a solvent containing HBTU and DIEA.

[0019] The invention discloses an antigen targeting peptide molecule, PEG2000-PLGLAG-preS1-pHLIP, wherein preS1 is SNNPDWDFNP, PLGLAG is an MMP2 restriction site, preS1 is an HBV antigen peptide, and the pHLIP low pH insertion peptide has a sequence of ACDDQNPWRAYDLLFPTDTLLLDLLW.

[0020] Preferably, in the preparation of the antigen targeting peptide molecule, PLGLAG-preS1-pHLIP and PEG polymer are mixed in DMF, and then HBTU and DIEA are added, and the reaction is shaken at 20-40° C. for 20-90 minutes to obtain PEG2000-PLGLAG-preS1-pHLIP. PLGLAG-preS1-pHLIP is synthesized sequentially from the C-terminus to the N-terminus of the sequence by solid phase synthesis.

[0021] More preferably, in the preparation of the antigen targeting peptide molecule, the PEG polymer includes PEG2000 or R-PEG, and the molar usage of the PEG polymer is 200-500% of the molar usage of PLGLAG-preS1-pHLIP.

[0022] More preferably, in the preparation of the antigen targeting peptide molecule, the molar amount of HBTU used is 200-500% of the molar amount of PLGLAG-preS1-pHLIP, and the molar amount of DIEA used is 900-1200% of the molar amount of PLGLAG-preS1-pHLIP.

[0023] More preferably, in the preparation of the antigen targeting peptide molecule, the usage amount of PLGLAG-preS1-pHLIP is 1-8wt% of DMF.

[0024] In the preparation of antigen targeting peptide molecules, PEG2000 can be replaced by R-PEG as the PEG polymer, and R-PEG-PLGLAG-preS1-pHLIP can be obtained.

[0025] The invention discloses an antigen targeting peptide molecule, R-PEG-PLGLAG-preS1-pHLIP, wherein preS1 is SNNPDWDFNP, PLGLAG is an MMP2 restriction site, preS1 is an HBV antigen peptide, and the pHLIP low pH insertion peptide has a sequence of ACDDQNPWRAYDLLFPTDTLLLDLLW.

[0026] The invention discloses a preparation method of R-PEG.

[0027] Preferably, in the preparation of R-PEG, a lactic acid aqueous solution and 5-hydroxynicotinic acid are mixed, and a prepolymer is prepared by dehydration under reduced pressure at a temperature of 130-165°C for 1-5h, and then PEG1500 is added, and then stannous chloride and p-toluenesulfonic acid are added, and then dehydration is carried out under reduced pressure at a temperature of 170-190°C for 2-10h, and then the reaction is carried out at a temperature of 130-165°C for 5-20h. After the reaction is completed, acetone is dissolved, and deionized water is precipitated and purified to obtain R-PEG.

[0028] More preferably, in the preparation of R-PEG, the lactic acid aqueous solution is prepared by mixing lactic acid and water, and the content of lactic acid in the lactic acid aqueous solution is 80-90wt%.

[0029] More preferably, in the preparation of R-PEG, the amount of 5-hydroxynicotinic acid used is 20-50 wt % of lactic acid.

[0030] More preferably, in the preparation of R-PEG, the amount of PEG1500 used is 20-50 wt % of lactic acid.

[0031] More preferably, in the preparation of R-PEG, the amount of stannous chloride used is 0.25-1.5wt% of the lactic acid, and the amount of p-toluenesulfonic acid used is 0.25-1.5wt% of the lactic acid.

[0032] In the preparation of antigen targeting peptide molecules, PLGLAG-preS1-pHLIP can be replaced with PLGLAG-Random-pHLIP, and PEG2000-PLGLAG-Random-pHLIP can be obtained, where Random is RGVPHIVMVD.

[0033] The invention discloses an antigen targeting peptide molecule, PEG2000-PLGLAG-Random-pHLIP, wherein Random is RGVPHIVMVD, PLGLAG is a MMP2 restriction site, and the pHLIP low pH insertion peptide has a sequence of ACDDQNPWRAYDLLFPTDTLLLDLLW.

[0034] The invention discloses a method for preparing antigen targeting peptide nano micelles. Antigen targeting peptide molecules are added into phosphate buffered saline, incubated at 20-40°C for 12-48h, and self-assembled to form antigen targeting peptide nano micelles.

[0035] Preferably, the phosphate buffered saline is a PBS solution with a pH of 6.5-7.8.

[0036] Preferably, the concentration of the antigen targeting peptide molecules added to phosphate buffered saline is 1.23-30 μM.

[0037] The antigen targeting peptide molecule disclosed in the present invention comprises at least one of PEG2000-PLGLAG-preS1-pHLIP, R-PEG-PLGLAG-preS1-pHLIP and PEG2000-PLGLAG-Random-pHLIP.

[0038] Preferably, when preparing antigen targeting peptide nano-micelles, antigen targeting peptide molecules and Cy5.5 can also be added to phosphate buffered saline for subsequent preparation, and Cy5.5 can be used in an appropriate amount.

[0039] Preferably, when preparing the antigen targeting peptide nano-micelles, the antigen targeting peptide molecules and sodium caseinate can also be added to phosphate buffered saline for subsequent preparation, and the amount of sodium caseinate used is 2-10wt% of the antigen targeting peptide molecules. When preparing the antigen targeting peptide nano-micelles of the present invention, sodium caseinate can also be added, and after the antigen targeting peptide nano-micelles are prepared by sodium caseinate and the antigen targeting peptide molecules of the present invention, the obtained antigen targeting peptide nano-micelles have enhanced therapeutic effects on tumors, and the antigen targeting peptide molecules containing R-PEG and sodium caseinate are used together, and the therapeutic effects on tumors are better.

[0040] The method for preparing the cell model with preS1 peptide inserted on the surface of the present invention further comprises treating the antigen targeting peptide nano-micelles with MPP2.

[0041] Preferably, in the treatment of antigen targeting peptide nanomicelles by MPP2, phosphate buffered saline is used to simulate the acidic TME environment, the pH is adjusted to 6.8, and the antigen targeting peptide nanomicelles and MMP2 are added for treatment for 1-12 hours. After the treatment is completed, the antigen targeting peptide nanomicelles treated with MPP2 are obtained.

[0042] More preferably, in the treatment of antigen targeting peptide nanomicelles by MPP2, the concentration of antigen targeting peptide nanomicelles is 10-30 μM.

[0043] More preferably, in the treatment of antigen targeting peptide nanomicelles with MPP2, the concentration of MMP2 is 0.1-0.5 μg / mL.

[0044] The present invention discloses a method for preparing a cell model with preS1 peptide inserted on the surface, wherein the antigen targeting peptide nano-micelles treated with MPP2 are mixed with SCC7 cells and incubated for 1-48 hours to prepare a cell model with preS1 peptide inserted on the surface. The antigen targeting peptide nano-micelles treated with MPP2 are used in an appropriate amount for incubation and culture.

[0045] The nano micelles of the present invention are composed of a hydrophilic terminal PEG polymer and a hydrophobic terminal fusion peptide. In the present invention, the PEG polymer can form an external protective hydrophilic layer during the self-assembly process, thereby effectively improving the water solubility of the polypeptide and significantly prolonging its blood circulation time in the body.

[0046] In the present invention, the MMP2 restriction site can achieve precise release in the TME. The preS1 peptide serves as a key epitope for antibody recognition and immune stimulation inside the tumor. pHLIP can anchor the preS1 epitope on the tumor cell membrane in the acidic TME.

[0047] In the present invention, the solid tumor is preferably head and neck squamous cell carcinoma.

[0048] The invention discloses use of an antigen targeting peptide molecule in preparing an anti-tumor drug.

[0049] The invention discloses use of antigen targeting peptide nano micelles in preparing anti-tumor drugs.

[0050] The invention adopts antigen targeting peptide molecules prepared by PEG polymer and fusion peptide of specific sequence, and then the antigen targeting peptide molecules are self-assembled in phosphate buffered saline to form antigen targeting peptide nano micelles, the PEG polymer at least includes PEG2000 and / or R-PEG, R is formed by prepolymerization of lactic acid and 5-hydroxynicotinic acid, and then R reacts with PEG to generate R-PEG, so it has the following beneficial effects: it can improve the immune microenvironment in the tumor, has good tumor targeting ability, activates antiviral immune memory after being released in the tumor microenvironment, can be inserted into the surface of tumor cells, has strong adaptability, avoids damage to normal cells and converts antiviral immunity into anti-tumor immunity. The antigen targeting peptide nano micelles of the invention can improve the survival rate of pre-immunized mice, and can improve the proportion of CD3+, CD3+CD4+ and CD3+CD8+T cells in the tumor of the pre-immunized mice. Therefore, the present invention is an antigen targeting peptide nano-micelle and its use which can improve the immune microenvironment in the tumor, has good tumor targeting ability, activates antiviral immune memory after being released in the tumor microenvironment, can be inserted into the surface of tumor cells, has strong adaptability, avoids damage to normal cells and converts antiviral immunity into anti-tumor immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the self-assembly and tumor killing strategy of antigen-targeted peptide nanomicelles.

[0052] Figure 2 Schematic diagram of electron microscopy characterization of antigen-targeting peptide nanomicelles.

[0053] Figure 3 Schematic diagram of electron microscopy characterization of antigen-targeting peptide nanomicelles after MPP2 treatment.

[0054] Figure 4 Schematic diagram of the in vitro functional evaluation of antigen-targeted peptide nanomicelles under low pH conditions.

[0055] Figure 5 Schematic diagram of the in vitro functional evaluation of antigen-targeted peptide nanomicelles on MMP2 and low pH conditions.

[0056] Figure 6 Schematic diagram of the “quenching-recovery” strategy for monitoring the release of nanomicelles in tumors.

[0057] Figure 7 Schematic diagram of the fluorescence kinetic results at different times for the in vivo experimental evaluation of the targeted release of antigen-targeted peptide nanomicelles in tumor tissues.

[0058] Figure 8 Schematic diagram of the fluorescence kinetic results of BHQ3-containing nanomicelles at different times for the in vivo evaluation of the targeted release of antigen-targeted peptide nanomicelles in tumor tissues.

[0059] Fig. 9 Schematic diagram of the fluorescence kinetics results of BHQ3-containing micelles sacrificed 12 hours later for in vivo experimental evaluation of targeted release of antigen-targeted peptide nanomicelles in tumor tissue.

[0060] Fig.10 Schematic representation of the pre-immunization plan for preS1 peptide.

[0061] Fig.11 Schematic diagram of the in vitro experimental evaluation results of the anti-tumor effects mediated by anti-preS1 antibodies and antigen-targeting peptide nanomicelles.

[0062] Fig.12 Schematic diagram of mouse tumor size.

[0063] Fig.13 Schematic diagram of Kaplan-Meier survival analysis curve.

[0064] Fig.14 CD3 + CD8 + Schematic diagram of T cell proportions. DETAILED DESCRIPTION

[0065] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0066] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0067] Example 1: An antigen targeting peptide molecule The antigen targeting peptide molecule is as follows: PEG2000-PLGLAG-preS1-pHLIP. preS1 is SNNPDWDFNP, which is a HBV antigen peptide. PLGLAG is the MMP2 restriction site, and the pHLIP low pH insertion peptide has the sequence ACDDQNPWRAYDLLFPTDTLLLDLLW.

[0068] Preparation of antigen targeting peptide molecules: PLGLAG-preS1-pHLIP and PEG polymer were mixed in DMF, and then HBTU and DIEA were added, and the reaction was shaken at 35°C for 45 minutes to obtain PEG2000-PLGLAG-preS1-pHLIP. PLGLAG-preS1-pHLIP was synthesized sequentially from the C-terminus to the N-terminus of the sequence by solid phase synthesis. The PEG polymer was PEG2000, the molar amount of the PEG polymer was 300% of the molar amount of PLGLAG-preS1-pHLIP, the molar amount of HBTU was 300% of the molar amount of PLGLAG-preS1-pHLIP, the molar amount of DIEA was 1000% of the molar amount of PLGLAG-preS1-pHLIP, and the amount of PLGLAG-preS1-pHLIP was 5wt% of DMF.

[0069] Example 2: A method for preparing antigen-targeting peptide nanomicelles, Preparation of antigen targeting peptide nano-micelles: Add antigen targeting peptide molecules to phosphate buffered saline, incubate at 35°C for 24 hours, and self-assemble to form antigen targeting peptide nano-micelles. Phosphate buffered saline is a PBS solution with a pH of 6.8; the concentration of the antigen targeting peptide molecules added to phosphate buffered saline is 10 μM. The antigen targeting peptide molecules are the antigen targeting peptide molecules in Example 1.

[0070] Example 3: A method for preparing antigen targeting peptide nanomicelles, The present embodiment is different from the embodiment 2 in that, in the preparation of the antigen targeting peptide nanomicelles, the antigen targeting peptide molecules and sodium caseinate are added to phosphate buffered saline, and the amount of sodium caseinate used is 5 wt % of the antigen targeting peptide molecules.

[0071] Example 4: Preparation method of a cell model with preS1 peptide inserted on the surface Treatment of antigen targeting peptide nanomicelles by MPP2: Phosphate buffered saline was used to simulate the acidic TME environment, the pH was adjusted to 6.8, and antigen targeting peptide nanomicelles and MMP2 were added for 4 hours. After the treatment was completed, antigen targeting peptide nanomicelles treated with MPP2 were obtained. The concentration of antigen targeting peptide nanomicelles was 20 μM, and the concentration of MMP2 was 0.2 μg / mL.

[0072] Preparation of a cell model with preS1 peptide inserted on its surface: The antigen-targeting peptide nanomicelles treated with MPP2 were mixed with SCC7 cells and incubated for 8 hours to prepare a cell model with preS1 peptide inserted on its surface.

[0073] Example 5: An antigen targeting peptide molecule Antigen targeting peptide molecules are shown below: R-PEG-PLGLAG-preS1-pHLIP. preS1 is SNNPDWDFNP, which is a HBV antigen peptide. PLGLAG is the MMP2 restriction site, and the pHLIP low pH insertion peptide has the sequence ACDDQNPWRAYDLLFPTDTLLLDLLW.

[0074] Preparation of R-PEG: Lactic acid aqueous solution and 5-hydroxynicotinic acid are mixed, and a prepolymer is prepared by decompression dehydration reaction at a temperature of 150°C for 3 hours, and then PEG1500 is added, and then stannous chloride and p-toluenesulfonic acid are added, and then decompression dehydration reaction is carried out at a temperature of 180°C for 6 hours, and then the reaction is carried out at a temperature of 150°C for 10 hours. After the reaction is completed, acetone is dissolved, and deionized water is precipitated and purified to obtain R-PEG. The lactic acid aqueous solution is prepared by mixing lactic acid and water, and the content of lactic acid in the lactic acid aqueous solution is 90wt%, the amount of 5-hydroxynicotinic acid used is 25wt% of lactic acid, the amount of PEG1500 used is 30wt% of lactic acid, the amount of stannous chloride used is 0.5wt% of lactic acid, and the amount of p-toluenesulfonic acid used is 0.5wt% of lactic acid.

[0075] Example 6: An antigen targeting peptide molecule Antigen targeting peptide molecules are shown below: R-PEG-PLGLAG-preS1-pHLIP. preS1 is SNNPDWDFNP, which is a HBV antigen peptide. PLGLAG is the MMP2 restriction site, and the pHLIP low pH insertion peptide has the sequence ACDDQNPWRAYDLLFPTDTLLLDLLW.

[0076] Preparation of R-PEG: Lactic acid aqueous solution and 5-hydroxynicotinic acid are mixed, and the prepolymer is prepared by decompression dehydration reaction at a temperature of 150°C for 3 hours, and then PEG1500 is added, and then stannous chloride and p-toluenesulfonic acid are added, and then decompression dehydration reaction is carried out at a temperature of 180°C for 6 hours, and then the reaction is carried out at a temperature of 150°C for 10 hours. After the reaction is completed, acetone is dissolved, and deionized water is precipitated and purified to obtain R-PEG. The lactic acid aqueous solution is prepared by mixing lactic acid and water, and the content of lactic acid in the lactic acid aqueous solution is 90wt%, the amount of 5-hydroxynicotinic acid used is 48wt% of lactic acid, the amount of PEG1500 used is 30wt% of lactic acid, the amount of stannous chloride used is 0.5wt% of lactic acid, and the amount of p-toluenesulfonic acid used is 0.5wt% of lactic acid.

[0077] Example 7: A method for preparing antigen-targeted peptide nanomicelles. The difference between this embodiment and embodiment 2 is that the antigen targeting peptide molecule is the antigen targeting peptide molecule in embodiment 5.

[0078] Example 8: A method for preparing antigen-targeted peptide nanomicelles. The difference between this embodiment and embodiment 2 is that the antigen targeting peptide molecule is the antigen targeting peptide molecule in embodiment 6.

[0079] Example 9: A method for preparing antigen-targeted peptide nanomicelles. The difference between this embodiment and embodiment 3 is that the antigen targeting peptide molecule is the antigen targeting peptide molecule in embodiment 5.

[0080] Example 10: A method for preparing antigen-targeted peptide nanomicelles. The difference between this embodiment and embodiment 3 is that the antigen targeting peptide molecule is the antigen targeting peptide molecule in embodiment 6.

[0081] Example 11: Preparation method of a cell model with preS1 peptide inserted on the surface The difference between this example and Example 4 is that the antigen targeting peptide nanomicelles used in this example are the antigen targeting peptide nanomicelles prepared in Example 7.

[0082] Example 12: Preparation method of a cell model with preS1 peptide inserted on the surface The difference between this example and Example 4 is that the antigen targeting peptide nanomicelles used in this example are the antigen targeting peptide nanomicelles prepared in Example 8.

[0083] Example 13: Preparation method of a cell model with preS1 peptide inserted on the surface The difference between this example and Example 4 is that the antigen targeting peptide nanomicelles used in this example are the antigen targeting peptide nanomicelles prepared in Example 9.

[0084] Example 14: Preparation method of a cell model with preS1 peptide inserted on the surface The difference between this example and Example 4 is that the antigen targeting peptide nanomicelles used in this example are the antigen targeting peptide nanomicelles prepared in Example 10.

[0085] Comparative Example 1: An antigen targeting peptide molecule The antigen targeting peptide molecule is as follows: PEG2000-PLGLAG-Random-pHLIP. Random is RGVPHIVMVD, PLGLAG is the MMP2 restriction site, and the pHLIP low pH insertion peptide has the sequence ACDDQNPWRAYDLLFPTDTLLLDLLW.

[0086] Comparative Example 2: An antigen targeting peptide molecule The antigen targeting peptide molecule is as follows: R-PEG-PLGLAG-preS1-pHLIP. preS1 is SNNPDWDFNP, which is an HBV antigen peptide. PLGLAG is an MMP2 restriction site, and pHLIP low pH insertion peptide, the sequence is ACDDQNPWRAYDLLFPTDTLLLDLLW. The preparation of R-PEG in this comparative example is compared with the preparation of R-PEG in Example 4, except that the amount of 5-hydroxynicotinic acid used is 30wt% of lactic acid.

[0087] Comparative Example 3: An antigen targeting peptide molecule The antigen targeting peptide molecule is as follows: R-PEG-PLGLAG-Random-pHLIP. Random is RGVPHIVMVD, PLGLAG is the MMP2 restriction site, and the pHLIP low pH insertion peptide has the sequence ACDDQNPWRAYDLLFPTDTLLLDLLW. The preparation of R-PEG in this comparative example is the same as that of R-PEG in Example 4.

[0088] Comparative Example 4: An antigen targeting peptide nanomicelle The difference between this comparative example and Example 2 is that the antigen targeting peptide molecule is the antigen targeting peptide molecule in Comparative Example 1.

[0089] Comparative Example 5: An antigen targeting peptide nanomicelle The difference between this comparative example and Example 2 is that the antigen targeting peptide molecule is the antigen targeting peptide molecule in Comparative Example 2.

[0090] Comparative Example 6: An antigen targeting peptide nanomicelle The difference between this comparative example and Example 2 is that the antigen targeting peptide molecule is the antigen targeting peptide molecule in Comparative Example 3.

[0091] Test example The self-assembly and use of the antigen targeting peptide nano-micelles prepared in Example 2 of the present invention are as follows: Figure 1 As shown, Figure 1 a in the figure is the antigen targeting peptide molecules self-assembled to form antigen targeting peptide nano-micelles. Figure 1 b in the figure is the in vivo tumor targeting mechanism of activating antiviral immune memory after the antigen-targeted peptide nanomicelles are released in the tumor microenvironment.

[0092] The present invention characterizes the antigen targeting peptide nanomicelles of Example 2, and the characterization results are as follows: Figure 2 As shown, Figure 2 a in is the particle size of antigen targeting peptide nanomicelles, Figure 2 b in the figure is a transmission electron micrograph of the antigen targeting peptide nanomicelles, wherein the nanomicelles are antigen targeting peptide nanomicelles.

[0093] In Example 4 of the present invention, MPP2 was used to treat the antigen targeting peptide nano-micelles. The characteristics of the antigen targeting peptide nano-micelles treated with MPP2 are as follows: Figure 3 As shown, Figure 3 a in the figure is the particle size diagram of antigen targeting peptide nanomicelles after MPP2 treatment. Figure 3 b is a transmission electron microscopy image of the antigen-targeting peptide nanomicelles after MPP2 treatment, wherein the nanomicelles are antigen-targeting peptide nanomicelles.

[0094] The present invention evaluates the in vitro reaction function of the antigen targeting peptide nanomicelles of Example 2 under low pH conditions, wherein the low pH conditions are selected to be pH 7.4 or 6.8, and preS1 in the SCC7 cell membrane is quantitatively detected by Western blot with or without MMP2 treatment. The results are as follows: Figure 4 As shown, S1: preS1 peptide, S2-S5: antigen-targeted peptide nanomicelles, β-actin as internal reference protein. SCC7 cells were incubated with PBS control, preS1 peptide or MMP2-treated and untreated antigen-targeted peptide nanomicelles at pH 7.4 or pH 6.8 for 2 h. Figure 4 a in the figure is the detection of preS1 peptide using anti-preS1 antibody. Figure 4b in which the preS1 peptide was detected using the serum of mice pre-immunized with preS1, and the membrane preS1-pHLIP was detected using anti-preS1 antibody or serum from mice pre-immunized with preS1(38-47), where preS1(38-47) is the preS1 peptide.

[0095] The in vitro response function of MMP2 to antigen-targeting peptide nanomicelles was evaluated under low pH conditions. Flow cytometry showed that SCC7 cells were exposed to MMP2-treated preS1-pHLIP nanomicelles at 37°C and pH 6.8. Figure 5 As shown, Figure 5 a in the figure is the change in the proportion of positive preS1 within 1, 2, 4, 8, 12 and 24 hours. Figure 5 b in the figure is the corresponding statistical analysis. Antigen targeting peptide nanomicelles were prepared as in Example 2.

[0096] The present invention loads Cyanine5.5 fluorophore (abbreviated as Cy5.5) and the corresponding distance-dependent quencher BHQ3 into nanomicelles, and indirectly monitors the dissociation of nanomicelles by measuring the fluorescence release of Cy5.5. The schematic diagram of the "quenching-recovery" strategy for monitoring the release of nanomicelles in tumors is shown in the figure. Figure 6 As shown. When preparing antigen targeting peptide nanomicelles, Cy5.5 and antigen targeting peptide nanomicelles are added together in phosphate buffered saline to form antigen targeting peptide @Cy5.5 nanomicelles. When preparing antigen targeting peptide nanomicelles, Cy5.5, BHQ3 and antigen targeting peptide nanomicelles are added together in phosphate buffered saline to form antigen targeting peptide @[Cy5.5+BHQ3] nanomicelles. Taking antigen targeting peptide @Cy5.5 nanomicelles as the baseline, the fluorescence quenching rate in antigen targeting peptide @[Cy5.5+BHQ3] nanomicelles reaches 73.8%. After the antigen targeting peptide @[Cy5.5+BHQ3] nanomicelles were treated with MMP2 for 2 hours, the quenching rate of the antigen targeting peptide @[Cy5.5+BHQ3] nanomicelles was reduced to 51.78%. The antigen targeting peptide nanomicelles were prepared in Example 2.

[0097] The present invention uses free Cy5.5-labeled preS1 peptide (abbreviated as Cy5.5-preS1 peptide) and compares it with the antigen targeting peptide @Cy5.5 nanomicelles of the present invention to verify the in vivo experiment of the antigen targeting peptide nanomicelles of the present invention in tumor tissue targeted release, and evaluates the tumor targeting of the nanomicelles after intravenous injection in the SCC7 BALB / c nude mouse subcutaneous tumor model. At 6 and 12 hours after injection, higher fluorescence density was observed at the tumor site using the antigen targeting peptide @Cy5.5 nanomicelles compared with the Cy5.5-preS1 peptide. Figure 7 As shown, Figure 7This is an in vivo experimental evaluation of the targeted release of antigen-targeted peptide nanomicelles in tumor tissues. After tail vein injection of PBS (Group 1, G1), Cy5.5-preS1 peptide (Group 2, G2) or antigen-targeted peptide@Cy5.5 nanomicelles (Group 3, G3) in SCC7 subcutaneous tumor-bearing mice, the statistical data of the fluorescence kinetic results were collected at 6, 12, 24 and 36 hours after injection, with a sample size of 3 mice per group (n=3); during long-term observation (24 and 36 hours after injection), although the metabolism of the peptide affected the fluorescence signal, the fluorescence intensity retained at the tumor site after nanomicelle delivery was still significantly higher than that of Cy5.5-preS1 peptide. The increased fluorescence density obtained by the nanomicelles at the tumor site may reflect its unique chemical properties and the prolonged in vivo circulation brought about by PEG modification. These results confirm the tumor targeting ability of the antigen-targeted peptide nanomicelles, which is of great significance for precision targeted therapy. The antigen-targeted peptide molecules in the antigen-targeted peptide nanomicelles are from Example 1.

[0098] The present invention uses the above-mentioned "quenching-recovery" probe to monitor the destruction of nanomicelles by TME rich in MMP2. Figure 8 As shown, Figure 8 This is an in vivo experimental evaluation of the targeted release of antigen-targeted peptide nanomicelles in tumor tissues. In SCC7 subcutaneous tumor-bearing mice, after tail vein injection of PBS (Group 1, A1), Cy5.5-preS1 peptide (Group 2, A2) or antigen-targeted peptide @[Cy5.5+BHQ3] nanomicelles (Group 3, A3), the statistical data of the fluorescence kinetic results were collected at 6, 12, 24 and 36 hours after injection, with a sample size of 4 mice per group (n=4); after injection of antigen-targeted peptide @[Cy5.5+BHQ3] nanomicelles, the released Cy5.5 fluorescent core was concentrated in the tumor site, and the highest fluorescence density reached a peak within 12 hours. This indicates that Cy5.5 is effectively dissociated and enriched at the tumor site. In addition, as Fig. 9 As shown, Fig. 9 This is an in vivo experimental evaluation of the targeted release of antigen-targeted peptide nanomicelles in tumor tissues. The fluorescence kinetic results of SCC7 subcutaneous tumor-bearing mice after intravenous injection of PBS (Group 1, A1), Cy5.5-preS1 peptide (Group 2, A2) or antigen-targeted peptide @[Cy5.5+BHQ3] nanomicelles (Group 3, A3) are statistical data. All mice were killed 12 hours after injection, and each group contained 4 mice; immunofluorescence analysis at different time points (i.e., 0, 2, 4, 6, 12 and 24 hours after injection of antigen-targeted peptide @[Cy5.5+BHQ3] nanomicelles) further showed that the antigen-targeted peptide nanomicelles of the present invention were significantly enriched in tumor tissues after 12 hours. These data show that the antigen-targeted peptide nanomicelles have good tumor targeting and release preS1-pHLIP under the action of TME. The antigen-targeted peptide molecules in the antigen-targeted peptide nanomicelles are from Example 1.

[0099] The presence of anti-preS1 antibodies is an important prerequisite for hepatitis B antigen-mediated tumor clearance. In order to evaluate the feasibility of using antigen-targeted peptide nanomicelles to modify B cell epitopes to promote antibody-mediated HNSCC immunotherapy, the present invention mixed preS1 peptides with alum adjuvant before tumor inoculation and immunized five-week-old immunocompetent C3H mice. Fig.10 As shown, mice received three intramuscular injections of preS1 peptide every two weeks. According to the ELISA test results, two weeks after the last injection (week 6), the average titer of anti-preS1 antibodies in the serum was 202.89 ng / μL, and remained stable in weeks 7 and 8. The antigen-targeting peptide nanomicelles were prepared in Example 2.

[0100] The present invention uses ADCC, CDC and ADCP to test the anti-tumor effect of anti-preS1 antibody in vitro. Fig.11 As shown, PreS1 is PreS1 peptide, Pre-pHLIP NMs is antigen-targeted peptide nanomicelles, and Pre-pHLIP NMs+MMP2 is antigen-targeted peptide nanomicelles+MMP2. In the ADCC experiment, NK cells were selected as effector cells, SCC7 cells were treated with treatment reagents as target cells, and specific cytotoxicity was determined by lactate dehydrogenase (LDH) release experiment. Fig.11 As shown in a, when SCC7 cells were pretreated with antigen-targeted peptide nanomicelles + MMP2, the ADCC induced by serum from mice immunized with preS1 peptide was significantly higher than that of the control group (PBS), antigen-targeted peptide nanomicelle group or the group treated with preS1 peptide only. The above-mentioned treatment reagents include PBS, preS1 peptide, antigen-targeted peptide nanomicelles and antigen-targeted peptide nanomicelles + MMP2. When SCC7 cells were pretreated with antigen-targeted peptide nanomicelles + MMP2, the ADCC induced by serum from mice immunized with preS1 was significantly higher than that of the PBS group, antigen-targeted peptide nanomicelle group and preS1 peptide group. In the CDC experiment, SCC7 cells were pretreated with the above-mentioned treatment reagents and incubated with serum collected at 6, 7 or 8 weeks after immunization. The group pretreated with antigen-targeted peptide nanomicelles + MMP2 had the lowest cell survival rate. As shown Fig.11 As shown in b, the results show that the group pretreated with antigen-targeted peptide nano-micelles + MMP2 has the lowest cell survival rate. Finally, the present invention uses fluorescent dye DIO to label SCC7 cells pretreated with the above treatment reagents, and co-cultures them with BMDMs and serum from preS1 immunized mice for 6 hours to measure ADCP. Fig.11As shown in c, the results of flow cytometry analysis showed that SCC7 cells treated with antigen targeting peptide nanomicelles + MMP2 had the highest phagocytic activity. These results indicate that preS1 antibodies bind to preS1 antigens on the surface of antigen targeting peptide nanomicelles + MMP2, which can effectively promote SCC7 tumors and participate in multiple mechanisms such as ADCC, CDC and ADCP. Antigen targeting peptide nanomicelles are prepared in Example 2.

[0101] In order to verify whether the antigen-targeted peptide nano-micelles can mobilize preS1 antibody-mediated humoral immunity and achieve tumor killing in vivo, five days after the last booster preS1 immunization, SCC7 cells were subcutaneously inoculated into C3H mice. Mice without pre-immunization were used as the control group and followed the same treatment regimen as the pre-immunization group. Specifically, the mice received five intravenous injections every four days, and the injection content included the antigen-targeted peptide nano-micelles of Comparative Example 2, preS1 peptide or the antigen-targeted peptide nano-micelles of Example 2. Fig.12 As shown, Group G1: no immunity, treated with PBS; Group G2: no immunity, treated with preS1 peptide; Group G3: no immunity, treated with the antigen-targeted peptide nano-micelles of Example 2; Group G4: PreS1 immunity, treated with PBS; Group G5: PreS1 immunity, treated with the antigen-targeted peptide nano-micelles of Comparative Example 4; Group G6: PreS1 immunity, treated with preS1 peptide; Group G7: PreS1 immunity, treated with the antigen-targeted peptide nano-micelles of Example 2; At the end of the treatment, mice that had not been pre-immunized with preS1 peptide did not show significant anti-tumor effects. Among the mice that had been pre-immunized, only the mice that received antigen-targeted peptide nano-micelle treatment showed significant anti-tumor activity. More importantly, in the pre-immunized group that received antigen-targeted peptide nano-micelle treatment, 70% of the mice were still alive after 60 days, while all the mice in other groups failed to survive, such as Fig.13As shown, Group G1: no immunity, treated with PBS; Group G2: no immunity, treated with preS1 peptide; Group G3: no immunity, treated with the antigen-targeted peptide nano-micelles of Example 2; Group G4: PreS1 immunity, treated with PBS; Group G5: PreS1 immunity, treated with the antigen-targeted peptide nano-micelles of Comparative Example 4; Group G6: PreS1 immunity, treated with preS1 peptide; Group G7: PreS1 immunity, treated with the antigen-targeted peptide nano-micelles of Example 2. These results indicate that the antigen-targeted peptide nano-micelles can exhibit significant anti-cancer activity in vivo by mobilizing existing anti-preS1 immunity. The unique inhibitory effect of the antigen-targeted peptide nano-micelles on HNSCC in pre-immunized mice strongly supports the strategy of tumor modification by inter-tumor release or transmembrane insertion of viral antigens to re-use antiviral immunity to promote anti-tumor immunity.

[0102] In order to characterize the effect of antigen-targeted peptide nano-micelles on the immune microenvironment in tumors, the immune cell components were analyzed by flow cytometry. Antigen-targeted peptide nano-micelle treatment promoted the expression of CD19 in pre-immunized animals. + and preS1+CD19 + Infiltration of B cells. Memory B cells are the cornerstone of protection against viral infection. An increase in the proportion of antigen-specific B cells may also indicate that the tumor has stronger antiviral immunity. In addition to B cells, antigen-targeted peptide nanomicelle treatment of pre-immunized mice increased intratumoral CD3 + 、CD3 + CD4 + and CD3 + CD8 + The proportion of T cells indicated that antiviral immunity also mobilized T cell immunity to participate in tumor killing. The increase in adaptive immune cells was accompanied by an increase in innate immune cells, including CD49 + NK cells, CD11C + Dendritic cells (DCs) and CD11B + 6G + Neutrophils. Antigen targeting peptide nanomicelles are prepared in Example 2.

[0103] The present invention used the antigen targeting peptide nano-micelles prepared in Examples 2-3, Examples 7-10 and Comparative Examples 4-6 to test the mice in the pre-immunized group. After the 20th day, CD3 + CD8 + T cell ratio Fig.14As shown, wherein, S2 is Example 2, S3 is Example 3, S7 is Example 7, S8 is Example 8, S9 is Example 9, S10 is Example 10, D4 is Comparative Example 4, D5 is Comparative Example 5, and D6 is Comparative Example 6. The present invention uses PEG polymers and fusion peptides of specific sequences to prepare antigen targeting peptide molecules, and then the antigen targeting peptide molecules are self-assembled in phosphate buffered saline to form antigen targeting peptide nano-micelles, the PEG polymer at least includes PEG2000 and / or R-PEG, R is pre-polymerized by lactic acid and 5-hydroxynicotinic acid, and then R is reacted with PEG to generate R-PEG, and the fusion peptide is PLGLAG-preS1-pHLIP. The antigen targeting peptide nano-micelles with a specific structure including PEG2000-PLGLAG-preS1-pHLIP or R-PEG-PLGLAG-preS1-pHLIP prepared by the above method of the present invention have good anti-tumor effects. After using the above antigen targeting peptide nano-micelles of the present invention to treat mice in the pre-immunized group, the CD3 + CD8 + The proportion of T cells in tumor cells is improved, while if the fusion peptide uses PLGLAG-Random-pHLIP, it cannot increase the CD3 + CD8 + The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention. The invention discloses a method for treating tumors of the present invention.

[0104] In addition, during the tumor killing process, tumor antigens are released and infiltrated into tumor tissues, and then presented to T cells by antigen-presenting cells to activate antigen-specific immune responses. Therefore, the present invention collected mouse spleen cells and stimulated them with tumor cell lysates overnight. The results of flow cytometry analysis showed that tumor antigen-specific T lymphocytes were significantly activated in pre-immunized mice after receiving antigen-targeted peptide nano-micelle treatment.

[0105] Overall, local release of preS1-pHLIP in tumors promoted TME remodeling by recruiting multiple immune cells from pre-immunized mice. In addition, combining antiviral antigens with antigen-targeting peptide nanomicelles and eliciting immune killing of tumor cells on the basis of pre-immunization may induce tumor antigen-specific adaptive immune responses.

[0106] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0107] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. An antigen targeting peptide molecule, as shown below: PEG polymer-PLGLAG-preS1-pHLIP, the PEG polymer includes any one of PEG and R-PEG, the molecular weight of the PEG is 1000-3000, the R has a lactic acid fragment and a 5-hydroxynicotinic acid fragment, and the molecular weight of the PEG in the R-PEG is 1000-3000.

2. An antigen targeting peptide molecule according to claim 1, characterized in that: The antigen targeting peptide molecule is as follows: PEG2000-PLGLAG-preS1-pHLIP; or, in the preparation of R-PEG, lactic acid and 5-hydroxynicotinic acid are pre-polymerized and then polymerized with PEG1500.

3. An antigen targeting peptide molecule according to claim 1, characterized in that: PLGLAG is an MMP2 restriction site, preS1 is a HBV antigen peptide, and pHLIP low pH insertion peptide has the sequence ACDDQNPWRAYDLLFPTDTLLLDLLW.

4. An antigen targeting peptide nanomicelle, comprising: The antigen targeting peptide molecule according to claim 1.

5. The antigen targeting peptide nanomicelle according to claim 4, characterized in that: The antigen targeting peptide nano-micelle is formed by self-assembly of antigen targeting peptide molecules in phosphate buffered saline.

6. The antigen targeting peptide nanomicelle according to claim 5, characterized in that: The pH of the phosphate buffered saline is 6.5-7.8; the concentration of the antigen targeting peptide molecules added to the phosphate buffered saline is 1.23-30 μM.

7. A method for preparing a cell model with preS1 peptide inserted on its surface, comprising: The antigen targeting peptide nanomicelles according to claim 4 are treated with MPP2, and then the antigen targeting peptide nanomicelles treated with MPP2 are mixed with SCC7 cells and incubated for 1-48 hours to prepare a cell model with preS1 peptide inserted on the surface.

8. A method for preparing an antigen targeting peptide molecule, comprising: The PEG polymer was prepared by reacting PLGLAG-preS1-pHLIP in a solvent containing HBTU and DIEA.

9. Use of an antigen targeting peptide molecule according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

10. Use of the antigen targeting peptide nanomicelle according to any one of claims 4 to 6 in the preparation of anti-tumor drugs.

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