Preparation of enzyme response engineered ferritin nano-particles and application of enzyme response engineered ferritin nano-particles in tumor combined therapy

Abstract

The invention discloses preparation of enzyme response engineered ferritin nanoparticles and application of the enzyme response engineered ferritin nanoparticles in tumor combined therapy. Coding sequences of PD-L1 blocking peptide, matrix metalloproteinase 2 / 9 substrate peptide and human heavy chain ferritin are connected in series by utilizing a genetic engineering technology, and recombinant ferritin (CMFn) is expressed by utilizing an escherichia coli prokaryotic expression system, so that the enzyme response recombinant ferritin with a PD-L1 blocking function is obtained, and the amino acid sequence is shown as SEQ ID NO.1. A chemotherapeutic drug oxaliplatin is further loaded in the inner cavity of the CMFn to form enzyme response functionalized ferritin nanoparticles, so that tumor targeted delivery and responsive release of an immune checkpoint inhibitor and the chemotherapeutic drug are realized, and the enzyme response functionalized ferritin nanoparticles have a stronger treatment effect and better biological safety, and can be used for preparing the immune checkpoint inhibitor and the chemotherapeutic drug in the tumor targeted delivery and the responsive release of the immune checkpoint inhibitor and the chemotherapeutic drug. The compound can be used for tumor targeted therapy or combined therapy.

Description

Technical Field

[0001] The present invention belongs to the field of nano biomedicine, and particularly relates to the preparation of an enzyme-responsive engineered ferritin nanoparticle and its application in tumor combination therapy. Background Art

[0002] Immune checkpoints play a crucial role in maintaining self-immune tolerance and regulating the duration and scope of peripheral tissue immune responses. However, these pathways can be "hijacked" by tumors and continuously activated, leading to tumor immune escape. Monoclonal antibody drugs targeting immune checkpoints have shown durable clinical responses in the treatment of various tumors.

[0003] Chemotherapy effectively enhances the therapeutic effect of immune checkpoint therapy and expands its indication range by enhancing tumor antigenicity and inducing tumor immunogenic death. However, chemotherapy drugs may cause a series of adverse reactions. In addition, the large molecular weight of monoclonal antibodies results in poor tumor permeability and certain immunogenicity. Immune-related adverse events caused by persistent target blockade and high costs further limit their application.

[0004] Polypeptides are considered potential alternative drugs to monoclonal antibodies. Compared with antibodies, polypeptides have lower manufacturing costs and immunogenicity, and stronger tumor penetrability. Currently, a variety of polypeptide inhibitors targeting immune checkpoints have been developed and achieved excellent therapeutic effects in preclinical studies. However, polypeptide drugs have problems such as low stability, short half-life, and low bioavailability. Nanotechnology has been widely studied in drug delivery, but due to complex synthesis processes, unknown long-term toxicity, etc., only a very small number of preparations can enter the clinical trial stage. In this context, endogenous self-assembling proteins with good biocompatibility are a better choice.

[0005] Ferritin is a natural iron storage protein with a spherical cage-like structure composed of 24 subunits. Ferritin has been widely used in the diagnosis and treatment of various diseases such as tumors due to its inherent tumor targeting, good biocompatibility, ease of production and modification, etc., and has the potential for clinical translation. Summary of the Invention

[0006] The primary object of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide an enzyme-responsive recombinant ferritin with PD-L1 blocking function.

[0007] Another object of the present invention is to provide a preparation method of the enzyme-responsive recombinant ferritin with PD-L1 blocking function.

[0008] Another object of the present invention is to provide the application of the enzyme-responsive recombinant ferritin with PD-L1 blocking function.

[0009] The object of the present invention is achieved by the following technical solutions:

[0010] An enzyme-responsive recombinant ferritin (CMFn) with PD-L1 blocking function, the amino acid sequence of which is shown in SEQ ID NO.1.

[0011] The enzyme-responsive recombinant ferritin is obtained by linking a PD-L1 blocking peptide, an MMP-2 / 9 substrate peptide, a flexible linker peptide and a human heavy chain ferritin subunit (from N-terminus to C-terminus).

[0012] The amino acid sequence of the PD-L1 blocking peptide is: WHRSYYTWNLNT (SEQ ID NO.7).

[0013] The amino acid sequence of the MMP-2 / 9 substrate peptide is: GPLGVR (SEQ ID NO.8).

[0014] The flexible linker peptide is: (GGGGS) n , where n is a natural number from 2 to 6 (preferably n = 1).

[0015] The amino acid sequence of the human heavy chain ferritin (HFn) is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0016] The coding gene of the enzyme-responsive recombinant ferritin (CMFn), the nucleotide sequence of which is shown in SEQ ID NO.2.

[0017] The preparation method of the enzyme-responsive recombinant ferritin with PD-L1 blocking function includes the following steps:

[0018] (1) The coding gene sequence of the recombinant ferritin (CMFn) shown in SEQ ID NO.2 and the pET-MBP-HRV3C expression plasmid are respectively double-digested with BamHⅠ and HindⅢ enzymes, and then ligated to obtain the CMFn expression plasmid;

[0019] (2) The CMFn expression plasmid is transformed into Escherichia coli competent cells, induced to express and purified to obtain the enzyme-responsive recombinant ferritin with PD-L1 blocking function (which can form a protein nanocage with a hollow spherical structure through self-assembly).

[0020] The Escherichia coli competent cells described in step (2) are preferably BL21 / DE3 E.coli competent cells.

[0021] The conditions for induced expression in step (2) are as follows: 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), cultured at 37 ± 1 °C for 6 - 10 hours (preferably 8 hours).

[0022] The purification in step (2) is carried out by Ni-IDA affinity chromatography column, and the eluent used is 300 mmol / L imidazole solution.

[0023] The application of the enzyme-responsive recombinant ferritin with PD-L1 blocking function in the preparation of PD-1 (immune checkpoint receptor programmed death receptor-1) blocker (inhibitor), anti-tumor drug carrier and / or anti-tumor drug.

[0024] The tumor mentioned above is a malignant tumor (most tumor cells highly express TfR1), including colon cancer, ovarian cancer, breast cancer, gastric cancer, pancreatic cancer, non-small cell lung cancer, melanoma, testicular tumor, lymphoma, head and neck tumor, etc.; preferably colon cancer.

[0025] The application of the combination of the enzyme-responsive recombinant ferritin with PD-L1 blocking function and chemotherapeutic drugs in the preparation of anti-tumor drugs.

[0026] The chemotherapeutic drug is preferably oxaliplatin.

[0027] The tumor mentioned above is a malignant tumor including colon cancer, ovarian cancer, breast cancer, gastric cancer, pancreatic cancer, non-small cell lung cancer, melanoma, testicular tumor, lymphoma, head and neck tumor, etc.; preferably colon cancer.

[0028] An enzyme-responsive engineered ferritin nanoparticle is obtained by encapsulating a chemotherapeutic drug with the above-mentioned enzyme-responsive recombinant ferritin with PD-L1 blocking function.

[0029] The chemotherapeutic drug is preferably oxaliplatin.

[0030] The preparation method of the enzyme-responsive engineered ferritin nanoparticle includes the following steps:

[0031] Add the above-mentioned enzyme-responsive recombinant ferritin (CMFn) with PD-L1 blocking function to a buffer solution with a pH of 2.5 ± 0.1, stir on ice, add the chemotherapeutic drug, and adjust the pH to 4.0 ± 0.1 to start the reassembly of the denatured ferritin. Then transfer the reaction system to a dialysis bag and dialyze it in a buffer solution with a pH of 7.4 at 4 °C to allow the ferritin to completely self-assemble, and at the same time encapsulate oxaliplatin in the inner cavity of the nanocage to obtain the enzyme-responsive engineered ferritin nanoparticle.

[0032] The buffer solution with a pH of 2.5±0.1 and the buffer solution with a pH of 7.4 are both 50 mmol / L Tris-HCl buffer solutions.

[0033] The chemotherapy drug is preferably oxaliplatin.

[0034] Adjusting the pH to 4.0±0.1 is preferably carried out using a 50 mmol / L Tris-HCl buffer solution with a pH of 9.2.

[0035] In the reaction system, the concentration of protein (CMFn) is 2 μmol / L, and the concentration of the chemotherapy drug is 4 mmol / L.

[0036] The cut-off molecular weight of the dialysis bag is 1 kD.

[0037] The dialysis time is 10 - 12 hours (preferably dialysis overnight).

[0038] The application of the enzyme-responsive engineered ferritin nanoparticles in the preparation of PD-1 blockers (inhibitors) and / or anti-tumor drugs.

[0039] The tumor is a malignant tumor including colon cancer, ovarian cancer, breast cancer, gastric cancer, pancreatic cancer, non-small cell lung cancer, melanoma, testicular tumor, lymphoma, head and neck tumors, etc.; preferably colon cancer.

[0040] The present invention has the following advantages and effects compared with the prior art:

[0041] 1. The present invention provides a preparation method of enzyme-responsive engineered ferritin nanoparticles and their application in tumor immuno-chemotherapy: The present invention uses genetic engineering technology to tandemly connect the coding sequences of PD-L1 blocking peptide, matrix metalloproteinase 2 / 9 (MMP-2 / 9) substrate peptide, and human heavy chain ferritin (HFn) (the amino acid composition of CMFn from the N-terminus to the C-terminus is: PD-L1 blocking peptide, MMP-2 substrate peptide, flexible linker peptide, HFn), and uses the Escherichia coli prokaryotic expression system to express recombinant ferritin (CMFn); further, the chemotherapy drug oxaliplatin (OXA) is loaded into the lumen of CMFn through pH-mediated disassembly / reassembly to form enzyme-responsive functionalized ferritin nanoparticles (CMFn@OXA) to achieve tumor-targeted delivery and responsive release of immune checkpoint inhibitors and chemotherapy drugs.

[0042] 2. The present invention provides an enzyme-responsive recombinant ferritin (CMFn) with PD-L1 blocking function. By means of genetic engineering technology, a PD-L1 blocking peptide is modified (fused) to the outer surface of human heavy chain ferritin (HFn), and linked with an MMP-2 / 9 substrate peptide (the purpose of using the MMP-2 substrate peptide is to enable CMFn to be cleaved by MMP-2 enzyme in the tumor microenvironment and release the PD-L1 blocking peptide). CMFn can self-assemble to form a protein nanocage with a hollow spherical structure. The introduced MMP-2 / 9 substrate peptide realizes the responsive release of the PD-L1 blocking peptide in the tumor microenvironment. The use of the Escherichia coli prokaryotic expression system can achieve the low-cost and large-scale preparation of recombinant ferritin. This recombinant ferritin (CMFn) can effectively block PD-L1 and enhance the anti-tumor immune response; this recombinant ferritin CMFn can be used for blocking PD-L1, restoring T cell activity, inhibiting tumor growth, and in tumor combination therapy. The present invention encapsulates oxaliplatin in the lumen of the recombinant protein CMFn, and the prepared CMFn@OXA has good tumor targeting and stronger anti-tumor efficacy compared with traditional combination therapies, realizing the co-delivery of immune checkpoint inhibitors and chemotherapeutic drugs.

[0043] 3. The preparation method of the enzyme-responsive engineered ferritin nanoparticles developed by the present invention can be used for the development of various stimulus-responsive ferritin drug delivery systems. The enzyme-responsive engineered ferritin nanoparticles developed based on this method have lower production costs, stronger therapeutic effects, and better biosafety than traditional combination therapies, and are a highly potential therapeutic strategy in future tumor combination therapy. Brief Description of the Drawings

[0044] Figure 1 is the plasmid map of recombinant ferritin; wherein, A is the plasmid map of MBP-CMFn; B is the plasmid map of MBP-CFn.

[0045] Figure 2 is the agarose gel electrophoresis detection result of the PCR product of recombinant ferritin; wherein, A is the agarose gel electrophoresis detection result of the PCR product of CMFn (in the figure, PCR1 is the product of the first PCR, and PCR2 is the product of the second PCR); B is the agarose gel electrophoresis detection result of the PCR product of CFn (in the figure, PCR1 is the product of the first PCR, and PCR2 is the product of the second PCR).

[0046] Figure 3It is the SDS-PAGE electrophoresis result diagram after the prokaryotic expression of HFn, MBP-CMFn, and MBP-CFn. (In the figure, lanes 1, 5, and 9 are the total proteins of the uninduced bacterial solution; lanes 2, 6, and 10 are the total proteins of the IPTG-induced bacterial solution; lanes 3, 7, and 11 are the precipitates of the bacterial cell lysates after IPTG induction; lanes 4, 8, and 12 are the supernatants of the bacterial cell lysates after IPTG induction).

[0047] Figure 4 It is the SDS-PAGE electrophoresis result diagram of the separation and purification of HFn, MBP-CMFn, and MBP-CFn. Among them, A is the SDS-PAGE analysis of HFn purified by anion exchange chromatography (In the figure, lane 1: sample flow-through; lane 2: 50 mM NaCl eluate; lane 3: 100 mM NaCl eluate; lane 4: 200 mM NaCl eluate; lane 5: 300 mM NaCl eluate; lane 6: 400 mM NaCl eluate; lane 7: 500 mM NaCl eluate; lane 8: 1 M NaCl eluate); B is the SDS-PAGE identification result of MBP-CMFn purified by Ni-IDA affinity chromatography (lane 1: sample flow-through; lane 2: 25 mM imidazole eluate; lane 3: 50 mM imidazole eluate; lane 4: 100 mM imidazole eluate; lane 5: 200 mM imidazole eluate; lane 6: 300 mM imidazole eluate); C is the SDS-PAGE identification result of MBP-CFn purified by Ni-IDA affinity chromatography (lane 1: total bacterial cell lysate; 2: supernatant of the bacterial cell lysate; lane 3: 100 mM imidazole eluate; lanes 4 - 6: 300 mM imidazole eluate).

[0048] Figure 5 It is the SDS-PAGE electrophoresis result diagram of MBP-CMFn and MBP-CFn after removing the tags and further purification. Among them, A is the SDS-PAGE analysis after digestion with HRV 3C enzyme (lane 1: MBP-CMFn digestion sample; lane 2: MBP-CFn digestion sample; the white arrow indicates CMFn and CFn after removing the tags); B is the SDS-PAGE analysis of purified HFn, CMFn, and CFn.

[0049] Figure 6 It is the size exclusion chromatography detection result diagram of HFn, CMFn, and CFn.

[0050] Figure 7 It is the transmission electron microscope image of HFn, CMFn, and CFn after negative staining.

[0051] Figure 8 It is the dynamic light scattering detection result diagram of HFn, CMFn, and CFn.

[0052] Figure 9 It is the Zeta potential detection result diagram of HFn, CMFn and CFn.

[0053] Figure 10 It is the circular dichroism detection result diagram of HFn, CMFn and CFn.

[0054] Figure 11 It is the result diagram of the change of particle size of HFn, CMFn and CFn in 10% FBS over time.

[0055] Figure 12 It is the flow cytometry detection result diagram of PD-L1 expression after treating MC38 cells with different concentrations of IFN-γ for 24 hours.

[0056] Figure 13 It is the immunofluorescence result diagram after co-incubating MC38 cells pretreated with IFN-γ with different drugs respectively.

[0057] Figure 14 It is the flow cytometry detection result diagram after co-incubating MC38 cells pretreated with IFN-γ with different drugs respectively; among them, A is the flow cytometry detection result; B is the mean fluorescence intensity.

[0058] Figure 15 It is the ELISA detection result diagram of the IFN-γ concentration in the supernatant after co-culturing MC38 cells pretreated with IFN-γ with CD8+ T cells for 48 hours in the presence of different drugs.

[0059] Figure 16 It is the cell viability detection result diagram of MC38 cells after co-culturing MC38 cells pretreated with IFN-γ with CD8+ T cells for 48 hours in the presence of different drugs.

[0060] Figure 17 It is the change diagram of the tumor volume of tumor-bearing mice during the treatment with CMFn in the MC38 mouse colon cancer model over time.

[0061] Figure 18 It is the tumor photo and tumor weight statistical chart of tumor-bearing mice after treatment with CMFn in the MC38 mouse colon cancer model; among them, A is the tumor photo; B is the tumor weight statistical chart.

[0062] Figure 19 It is the change diagram of the body weight of tumor-bearing mice during the treatment with CMFn in the MC38 mouse colon cancer model over time.

[0063] Figure 20 It is the flow cytometry analysis result diagram of immune cells in the tumor tissue of tumor-bearing mice after treatment with CMFn in the MC38 mouse colon cancer model.

[0064] Figure 21 are negative staining transmission electron microscopy images during the pH-mediated depolymerization-reassembly process for encapsulating OXA; among them, A is the negative staining transmission electron microscopy image of CMFn under the condition of pH 2.5; B is the negative staining transmission electron microscopy image of CMFn@OXA after encapsulation is completed.

[0065] Figure 22 is the detection result graph of the standard curve of OXA; among them, A is the ultraviolet-visible light spectrum graph of OXA with different concentrations; B is the standard curve graph of OXA concentration and absorbance.

[0066] Figure 23 are the ultraviolet-visible light spectrum graphs of OXA, CMFn, and CMFn@OXA.

[0067] Figure 24 is the drug release curve graph of OXA in HFn@OXA and CMFn@OXA under different pH conditions.

[0068] Figure 25 is the change graph of the remaining percentage of OXA in CMFn@OXA over time in PBS and FBS.

[0069] Figure 26 are the detection result graphs of cell viability after treating MC38 cells, HTC116 cells, and HUVEC cells with different OXA concentrations for 72 hours respectively.

[0070] Figure 27 are the CLSM images after incubating Cy5.5-labeled CMFn@OXA with HCT116 cells for different times (in the figure, red is Cy5.5-labeled CMFn@OXA, and blue is the cell nucleus).

[0071] Figure 28 are the CLSM images after incubating Cy5.5-labeled CMFn@OXA with HCT116 cells for 12 hours (in the figure, red is Cy5.5-labeled CMFn@OXA, green is lysosome, and blue is the cell nucleus).

[0072] Figure 29 is the graph of the distribution of the drug in mice over time.

[0073] Figure 30 are the fluorescence imaging graphs of tumors, heart, liver, spleen, lung, and kidney of mice.

[0074] Figure 31 is the graph of the change in tumor volume of tumor-bearing mice over time during the treatment of MC38 mouse colon cancer model with CMFn@OXA.

[0075] Figure 32It is a statistical chart of tumor photos and tumor weights of tumor-bearing mice after treatment with CMFn@OXA in the MC38 mouse colon cancer model; among them, A is the tumor photo; B is the statistical chart of tumor weights.

[0076] Figure 33 It is a graph showing the change in the body weight of tumor-bearing mice over time during the treatment with CMFn@OXA in the MC38 mouse colon cancer model.

[0077] Figure 34 It is a survival curve of tumor-bearing mice after treatment with CMFn@OXA in the MC38 mouse colon cancer model.

[0078] Figure 35 It is a graph of the ELISA test results of the concentrations of IFN-γ and TNF-α in the tumor tissues of tumor-bearing mice after treatment with CMFn@OXA in the MC38 mouse colon cancer model; among them, A is IFN-γ; B is TNF-α.

[0079] Figure 36 It is an immunofluorescence staining map of tumor tissue sections of tumor-bearing mice after treatment with CMFn@OXA in the MC38 mouse colon cancer model (in the figure, green is CD4, red is CD8, and blue is the cell nucleus).

[0080] Figure 37 It is a graph of the HE staining results of the main organs of tumor-bearing mice after treatment with CMFn@OXA in the MC38 mouse colon cancer model.

[0081] Figure 38 It is a graph of the serum biochemical test results of tumor-bearing mice after treatment with CMFn@OXA in the MC38 mouse colon cancer model. Detailed implementation manners

[0082] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. The test methods without specific experimental conditions in the following embodiments are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained through commercial channels.

[0083] Example 1 Preparation and characterization of CMFn and its mutant CFn

[0084] 1. Materials and reagents

[0085] Preparation of buffer solution:

[0086] Buffer A: 50 mM Tris-HCl pH 8.0, 1 mM phenylmethylsulfonyl fluoride (PMSF);

[0087] Buffer B: 50 mM Tris-HCl pH 8.0, 100 mM NaCl;

[0088] Buffer C: 50 mM Tris-HCl pH 8.0, 300 mM NaCl;

[0089] Buffer D: 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole;

[0090] Buffer E: 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 100 mM imidazole;

[0091] Buffer F: 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 300 mM imidazole.

[0092] 2. Method

[0093] 2.1 Construction of CMFn and CFn expression vectors

[0094] According to the amino acid sequences of the PD-L1 blocking peptide, MMP-2 substrate peptide, and flexible linker peptide (PD-L1 blocking peptide: WHRSYYTWNLNT; MMP-2 substrate peptide: GPLGVR; flexible linker peptide: GGGGS), codon optimization for Escherichia coli was carried out. Using the ferritin expression vector pET30a(+)-HFn (purchased from Sangon Biotech) as a template, primer pairs containing the coding sequences of the PD-L1 blocking peptide, MMP-2 substrate peptide, flexible linker peptide, and restriction enzyme sites were designed to perform two rounds of PCR amplification on the ferritin fragment (aa5-aa174) (protein sequence as shown in SEQ ID NO.3) respectively, to obtain the CMFn fragment (protein sequence as shown in SEQ ID NO.1) and the CFn fragment (protein sequence as shown in SEQ ID NO.5). The upstream primers all carried the coding sequences of the PD-L1 blocking peptide, MMP-2 substrate peptide, and flexible linker peptide and the BamHⅠ restriction enzyme site, and the downstream primers all carried the HindⅢ restriction enzyme site. The PCR results were identified by agarose gel electrophoresis. The PCR products were purified using a gel extraction kit. The purified products and the pET-MBP-HRV 3C expression plasmid (purchased from Sangon Biotech) were digested with two restriction enzymes respectively, and ligated overnight at 16 °C under the action of T4 ligase according to the molar ratio of vector:product of 1:10. The ligation products were transformed into DH5α E. coli competent cells to screen for correctly ligated clones, and the successfully constructed CMFn and CFn plasmids were obtained. Among them,

[0095] The amplification primers for the first PCR of CMFn are:

[0096] CMFn-PCR1-F: 5′-GAACACTGGTGGTCCGCTGGGCGTTCGTGGCGGCGGCACGACCGCGTCCACC-3′;

[0097] CMFn-PCR1-R: 5′-CCCAAGCTTCTAGCTTTCATTATCACTGTCTCCCAGG-3′;

[0098] The amplification primers for the second PCR of CMFn are:

[0099] CMFn-PCR2-F: 5′-CGGGATCCTGGCATCGTTCTTACTACACGTGGAATCTGAACACTGGTGGTCCGCT-3′;

[0100] CMFn-PCR2-R: 5′-CCCAAGCTTCTAGCTTTCATTATCACTG-3′;

[0101] The amplification primers for the first PCR of CFn are:

[0102] CFn-PCR1-F: 5′-GAACACTGGTGGTGGCTCTGGTGGTTCTGGCGGCGGCACGACCGCGTCCACC-3′; CFn-PCR1-R: 5′-CCCAAGCTTCTAGCTTTCATTATCACTGTCTCCCAGG-3′;

[0103] The amplification primers for the second PCR of CFn are:

[0104] CFn-PCR2-F: 5′-CGGGATCCTGGCATCGTTCTTACTACACGTGGAATCTGAACACTGGTGGTGGCTCTG-3′;

[0105] CFn-PCR2-R: 5′-CCCAAGCTTCTAGCTTTCATTATCACTG-3′.

[0106] The amino acid sequence of ferritin (HFn) (SEQ ID NO.3):

[0107] MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREH AEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIE THYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES。

[0108] Ferritin (HFn) nucleotide sequence (SEQ ID NO.4):

[0109] atgaccaccgcgagcaccagccaggtgcgccagaactatcatcaggatagcgaagcggcgattaaccgccagattaacctggaactgtatgcgagctatgtgtatctgagcatgagctattattttgatcgcgatgatgtggcgctgaaaaactttgcgaaatattttctgcatcagagccatgaagaacgcgaacatgcggaaaaactgatgaaactgcagaaccagcgcggcggccgcatttttctgcaggatattaaaaaaccggattgcgatgattgggaaagcggcctgaacgcgatggaatgcgcgctgcatctggaaaaaaacgtgaaccagagcctgctggaactgcataaactggcgaccgataaaaacgatccgcatctgtgcgattttattgaaacccattatctgaacgaacaggtgaaagcgattaaagaactgggcgatcatgtgaccaacctgcgcaaaatgggcgcgccggaaagcggcctggcggaatatctgtttgataaacataccctgggcgatagcgataacgaaagc。

[0110] CMFn amino acid sequence (SEQ ID NO.1):

[0111] WHRSYYTWNLNTGGPLGVRGGGTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES。

[0112] CMFn nucleotide sequence (SEQ ID NO.2):

[0113] tggcatcgcagctattatacctggaacctgaacaccggcggcccgctgggcgtgcgcggcggcggcaccaccgcgagcaccagccaggtgcgccagaactatcatcaggatagcgaagcggcgattaaccgccagattaacctggaactgtatgcgagctatgtgtatctgagcatgagctattattttgatcgcgatgatgtggcgctgaaaaactttgcgaaatattttctgcatcagagccatgaagaacgcgaacatgcggaaaaactgatgaaactgcagaaccagcgcggcggccgcatttttctgcaggatattaaaaaaccggattgcgatgattgggaaagcggcctgaacgcgatggaatgcgcgctgcatctggaaaaaaacgtgaaccagagcctgctggaactgcataaactggcgaccgataaaaacgatccgcatctgtgcgattttattgaaacccattatctgaacgaacaggtgaaagcgattaaagaactgggcgatcatgtgaccaacctgcgcaaaatgggcgcgccggaaagcggcctggcggaatatctgtttgataaacataccctgggcgatagcgataacgaaagc。

[0114] CFn amino acid sequence (SEQ ID NO.5):

[0115] WHRSYYTWNLNTGGGSGGSGGGTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES。

[0116] CFn nucleotide sequence (SEQ ID NO.6):

[0117] tggcatcgcagctattatacctggaacctgaacaccggcggcggcagcggcggcagcggcggcggcaccaccgcgagcaccagccaggtgcgccagaactatcatcaggatagcgaagcggcgattaaccgccagattaacctggaactgtatgcgagctatgtgtatctgagcatgagctattattttgatcgcgatgatgtggcgctgaaaaactttgcgaaatattttctgcatcagagccatgaagaacgcgaacatgcggaaaaactgatgaaactgcagaaccagcgcggcggccgcatttttctgcaggatattaaaaaaccggattgcgatgattgggaaagcggcctgaacgcgatggaatgcgcgctgcatctggaaaaaaacgtgaaccagagcctgctggaactgcataaactggcgaccgataaaaacgatccgcatctgtgcgattttattgaaacccattatctgaacgaacaggtgaaagcgattaaagaactgggcgatcatgtgaccaacctgcgcaaaatgggcgcgccggaaagcg gcctggcggaatatctgtttgataaacataccctgggcgatagcgataacgaaagc。

[0118] 2.2 Expression of HFn, CMFn and CFn

[0119] The pET30a(+)-HFn plasmid and the successfully constructed CMFn and CFn plasmids were respectively transformed into BL21(DE3) E. coli competent cells, heat-activated at 42 °C for 90 s, and spread on an ampicillin (Amp + )(final concentration 50 μg / ml) resistant LB solid culture plate. Positive clones were picked and cultured overnight at 37 °C with 220 rpm in 5 mL of LB (Amp + , final concentration 50 μg / ml) liquid medium. The bacterial solution was inoculated into 2 L of LB (Amp + , final concentration 50 μg / ml) liquid medium at a ratio of 1:1000 (volume ratio) and cultured under the same conditions for 3 - 4 h. At this time, the OD 600 was approximately 0.6. Then, isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.5 mM was added and cultured at 37 °C for 8 hours to induce the expression of the target protein. The cells were collected by centrifugation at 4500 rpm for 20 minutes. HFn was resuspended in buffer A, and then the cells were disrupted using a high-pressure homogenizer to release the target protein. The cell debris was removed by centrifugation at 12000 rpm for 20 minutes. The supernatant was filtered through a 0.22 μM syringe filter and stored at 4 °C for later use.

[0120] 2.3 Purification of HFn, CMFn, and CFn

[0121] HFn was initially purified by heat treatment and anion exchange chromatography. First, the supernatant of the cell lysate was heated in a water bath at 70 °C for 15 minutes to preliminarily remove miscellaneous proteins using the thermal stability of ferritin. After centrifugation at 12000 rpm for 10 minutes, the supernatant was taken and passed through a 0.22 μM filter membrane. The chromatography column was clamped on an upright support, and then Q-Sepharose FF was packed into the chromatography column. A glass rod was used to drain along the inner wall of the column closely to reduce the generation of bubbles. The gel was allowed to settle freely in the column, and the column was rinsed twice with distilled water to remove residual ethanol, and then buffer A was added for column equilibration. The sample obtained in the previous step was added to the chromatography column, and the flow-through was collected. The loaded chromatography column was rinsed with 4 - 5 column volumes of buffer A to remove unbound proteins. Elution buffers of 50, 100, 200, 300, 400, and 500 mM NaCl were respectively prepared to elute the target protein. The chromatography column was rinsed with 8 - 10 column volumes of buffer B (containing 100 mM NaCl) to remove weakly bound miscellaneous proteins, and then the target protein was eluted with 4 - 5 column volumes of buffer C.

[0122] The CMFn (MBP-CMFn) and CFn (MBP-CFn) of the MBP tag were purified by Ni-IDA affinity chromatography column. First, Ni-IDA Sepharose FF was packed into the chromatography column, and air bubbles were avoided during the packing process. The packing material was allowed to settle naturally under the action of gravity, and the column was rinsed twice with distilled water to remove residual ethanol, and then equilibrated with 2-3 column volumes of buffer D. NaCl with a final concentration of 500 mM was added to the supernatant of the cell lysate to reduce the influence of ion exchange, and imidazole with a final concentration of 20 mM was added to reduce non-specific binding. Then, after filtration through a 0.22 μM filter membrane, it was added to the chromatography column, and the flow-through was collected. The chromatography column was rinsed with 4-5 column volumes of buffer D (containing 20 mM imidazole) to remove unbound proteins. The chromatography column was rinsed with 8-10 column volumes of buffer E (containing 100 mM imidazole) to remove weakly bound impurities, and the target protein was eluted with 4-5 column volumes of buffer F (containing 25, 50, 100, 200, 300 mM imidazole). Then, the imidazole in the protein was removed by dialysis, the protein concentration was detected by the BCA method, and the protein was diluted to an appropriate concentration. HRV 3C protease (purchased from Sangon Biotech) was added and digested at 4°C for 16 hours to remove the MBP tag. After digestion, it was passed through the Ni-IDA affinity chromatography column again to remove the MBP tag and HRC 3C protease, and the flow-through was collected.

[0123] The protein obtained through the above steps was further purified and identified using size exclusion chromatography. First, the preliminarily purified protein was concentrated using an ultrafiltration concentrator tube (cut-off molecular weight of 10 kD), and then loaded into a dialysis bag (cut-off molecular weight of 10 kD) and dialyzed in PBS at 4°C for 24 hours to replace the buffer of the protein sample with PBS. All samples and buffers were passed through a 0.22 μM filter membrane before loading onto the column to remove possible particles. The column was rinsed with ddH2O, and then flushed forward with 0.5 M NaOH at a flow rate of 3 mL / min for 3 column volumes. It was rinsed with ddH2O for 3 column volumes, and then rinsed with PBS for 5-10 column volumes to equilibrate the column. Then, the concentrated sample was injected into the chromatography column, and the loading volume was 0.5 mL. After loading, it was eluted with PBS at a flow rate of 1.5 mL / min, and the eluate was collected in 3 mL tubes. After purification, it was flushed backward with 0.5 M NaOH for 2 column volumes for 30-60 minutes. After flushing, the column was rinsed with 5 column volumes of ddH2O, and finally rinsed with 20% ethanol for 3 column volumes.

[0124] 2.1 Characterization of HFn, CMFn and CFn

[0125] The morphology of protein cages (HFn, CMFn, and CFn) was characterized using a transmission electron microscope (TEM). The sample was centrifuged at 12000 rpm for 10 minutes, and the supernatant was taken and filtered through a 0.22 μm syringe filter, then diluted to an appropriate concentration with PBS; 5 μL of the sample suspension was directly dropped onto a copper grid with a carbon support film and left standing for 3 - 5 minutes; the excess liquid was blotted away from the edge of the droplet with a filter paper and slightly dried; 2 μL of 3% phosphotungstic acid negative staining solution was added and left standing for 2 - 3 minutes; the excess staining solution was blotted away and observed under a transmission electron microscope after natural drying.

[0126] The particle size and Zeta potential of the protein cages were measured using a nanoparticle size analyzer. First, the sample was diluted to an appropriate concentration with PBS, the instrument was set according to the manufacturer's instructions, the sample was loaded into the sample cell, air bubbles were removed, the sample cell was placed in the instrument and the measurement was started. Each sample was measured three times and the average value was taken, and 10 cycles were measured each time.

[0127] The circular dichroism spectrum of the protein was detected using a circular dichroism spectrometer. According to the manufacturer's instructions, first, the switch of the nitrogen gas cylinder was opened and the air pressure was adjusted to 0.4 MPa. The instrument and software were turned on, and nitrogen purging was carried out for 20 minutes. The xenon light source was turned on, the spectral measurement range was set to 190 nm - 250 nm, the bandwidth was 1 nm, the step was 1 nm, and the sampling time was 0.5 s. First, the air background was measured, and then PBS was loaded into the cuvette to measure the baseline. The sample was diluted to 200 μg / mL, loaded into the cuvette for sample measurement, and each sample was measured three times repeatedly.

[0128] Serum stability analysis. First, the sample was filtered through a 0.22 μM syringe filter and diluted to an appropriate concentration with PBS, then sterile FBS with a final concentration of 10% (v / v) was added, sealed with a sealing film and stored at 4°C. A part of the sample was taken every 24 hours for particle size measurement. The experiment was set up with three replicates.

[0129] 3. Results:

[0130] The expression plasmid maps of CMFn and CFn are as Figure 1 shown. The agarose gel electrophoresis results of the PCR products are as Figure 2 shown. The DNA bands are clear and no obvious miscellaneous bands appear, indicating a high amplification efficiency and strong primer specificity. In addition, the band position is below 1 kb, which is consistent with the theoretical size (546 bp) of the target fragment.

[0131] The results of SDS - PAGE are as Figure 3As shown, after IPTG induction, obvious protein bands appeared at the corresponding positions (white boxes) of the three target proteins, indicating the successful induction of the expression of the target proteins. In addition, the bands of HFn, MBP-CMFn, and MBP-CFn appeared in the supernatant of the cell lysate, indicating their expression in a soluble form. According to the properties of the target proteins, we adopted different methods for separation and purification. SDS-PAGE analysis of the purification process of HFn showed that HFn was eluted at an ionic strength of 300 mM NaCl.

[0132] SDS-PAGE analysis of the purification processes of MBP-CMFn and MBP-CFn is as Figure 4 shown. These two proteins have similar nickel column binding abilities and are both eluted in large amounts at an imidazole concentration of 300 mM. To exclude the effects of the MBP and His tags on the structures and functions of CMFn and CFn, we digested the target proteins with HRV 3C protease. The further purification results are as Figure 5 shown. After digestion, two protein bands appeared. The band at 40 kD to 50 kD was the MBP tag (theoretical molecular weight is about 43 kD), while the bands of CMFn and CFn (theoretical molecular weight is about 23.7 kD) appeared at around 25 kD. Since both the MBP tag and HRV 3C protease carry His tags, the digestion products were further purified and concentrated by nickel column and size exclusion chromatography. The purities of the three finally purified proteins can all reach over 95%, and CMFn and CFn have a larger molecular weight than HFn. To study whether polypeptide modification would affect the self-assembly and structure of ferritin, we characterized the properties such as the morphology, particle size, and Zeta potential of HFn, CFMn, and CFn.

[0133] The size exclusion chromatography (SEC) results of HFn, CMFn, and CFn are as Figure 6 shown. The elution volumes of CMFn and CFn are smaller than that of HFn, indicating that the migration speeds of CMFn and CFn in the chromatographic column are faster than that of HFn. Since the migration speed of the sample is positively correlated with the molecular size, it indicates that CMFn and CFn have a larger molecular weight or volume than HFn. Considering that HFn can self-assemble into 24-mers in the native state, this suggests that CMFn and CFn may also self-assemble into multimers with a larger molecular weight.

[0134] The results of TEM are as Figure 7As shown, HFn can self-assemble to form spherical cage-like structures with a particle size of about 12 nm. Similar to HFn, CMFn and CFn are also spherical with uniform size and uniform dispersion, and their particle sizes are slightly larger than that of HFn. A black core appears inside some particles, which may be due to the penetration of phosphotungstic acid stain through the channels between ferritin subunits into the inner cavity during the negative staining process. The results of TEM show that the recombinant ferritin fused with polypeptide and natural ferritin have similar morphological characteristics, indicating that ferritin can still self-assemble into spherical structures after polypeptide modification.

[0135] The particle sizes of HFn, CMFn and CFn in PBS were measured by dynamic light scattering (DLS) respectively. DLS is a mature non-invasive technique that can measure the particle size and particle size distribution of molecules and particles in the submicron range. The measurement results are as Figure 8 shown. The particle size distributions of the three kinds of nanocages are relatively concentrated. Among them, the particle size distribution of HFn is 11.9 ± 0.5 nm, which is consistent with the literature reports. After fusing with polypeptide, the particle sizes of CMFn and CFn increase to 20.6 ± 0.5 nm and 21.7 ± 0.9 nm respectively.

[0136] We measured the Zeta potentials of HFn, CMFn and CFn in PBS respectively, and the results are as Figure 9 shown. The Zeta potentials of HFn, CMFn and CFn are -11.7 ± 0.4 mV, -18.5 ± 0.4 mV and -18.5 ± 0.2 mV respectively, indicating that they are all negatively charged in neutral PBS, and the absolute values of the potentials of CMFn and CFn are larger than that of HFn, which suggests that CMFn and CFn may have better dispersibility and stability.

[0137] The circular dichroism spectra of HFn, CMFn and CFn are as Figure 10 shown. The results show that polypeptide modification has no obvious effect on the secondary structure of ferritin. In addition, to test the stability of the nanocages, we used 10% (v / v) fetal bovine serum (FBS) to simulate the plasma environment and detected the particle size changes of HFn, CMFn and CFn in serum. The test was carried out once a day for 7 days. The experiment was set up with three replicates. The results are as Figure 11 shown. During the 7-day test, the particle sizes of these three kinds of nanocages in FBS did not change significantly, indicating that they all have good stability.

[0138] Example 2 In vitro biological blood effect study of enzyme-responsive recombinant ferritin

[0139] 1. Seed MC38 cells (purchased from ATCC) in a 6-well plate and incubate overnight at 37 °C to allow the cells to adhere. Then, add IFN-γ (Proteintech) at a final concentration of 25 ng / mL or 50 ng / mL and treat for 24 hours. After the treatment, collect the cells and perform cell counting. Transfer 5×10 5 cells from each sample to a 1.5 mL centrifuge tube for subsequent staining. Centrifuge at 100 g for 3 minutes to collect the cells, resuspend them in 50 μL of CD16 / 32 antibody (BioLegend) dilution (10 μg / mL), and incubate on ice for 20 minutes. Then, add 50 μL of PE-CD274 antibody (BioLegend) dilution (2.5 μg / mL) to each tube and mix well, and incubate protected from light on ice for 30 minutes. After the incubation, add 1 mL of PBS to each tube to wash away the unbound antibodies, centrifuge at 450 g for 5 minutes at 4 °C, discard the supernatant, and repeat 3 times. Resuspend the cells in 300 μL of PBS in each tube, transfer to a flow cytometry tube, and detect using a flow cytometer. Use PBS as a control. The experiment was set up with three replicates. The results are as Figure 12 shown. IFN-γ can effectively upregulate the expression of PD-L1 in MC38 cells, and the expression level of PD-L1 increases with the increase in the concentration of IFN-γ.

[0140] 2. Inoculate MC38 cells in the logarithmic growth phase into a 35-mm glass-bottom confocal culture dish, culture overnight at 37 °C to allow the cells to adhere, then add IFN-γ (50 ng / mL) and treat for 24 hours. Then, add HFn, CMFn, CFn prepared in Example 1, and the anti-PD-L1 short peptide inhibitor CLP002 (Genepept Biotech) (equivalent to the amount of polypeptide at 0.1 mg / mL, that is, calculated by polypeptide, the final concentration is 0.1 mg / mL) and continue to culture for 4 hours. After the treatment, aspirate the culture medium, wash twice with PBS, add 4% paraformaldehyde and fix at room temperature for 10 minutes, wash 3 times with PBS, 5 minutes each time. Add PBS buffer containing 0.2% (v / v) Triton X-100 and permeabilize at room temperature for 5 minutes, wash 3 times with PBS, 5 minutes each time. Add 5% (v / v) goat serum and block at room temperature for 30 minutes. Aspirate the blocking solution, add the dilution of PD-L1 antibody (Proteintech) (1:300, v / v), incubate overnight at 4 °C, wash 3 times with PBS, 5 minutes each time. Add the dilution of Alexa Fluor 647 anti-mouse IgG1 antibody (BioLegend) (2.5 μg / mL), place on a shaker and incubate at room temperature in the dark for 1 hour, wash 3 times with PBS, 5 minutes each time. Add DAPI staining solution (0.5 μg / mL), incubate at room temperature in the dark for 15 minutes, wash 3 times with PBS, 5 minutes each time. After the washing is completed, observe using a fluorescence microscope. Use PBS as a control. The experiment is set up with three replicates. The results are as Figure 13 shown. Compared with the HFn control group, almost no PD-L1 fluorescence signal was observed in the CMFn and CLP002 experimental groups, indicating that they can block PD-L1 and inhibit the binding of the PD-L1 antibody to PD-L1. In contrast, a certain fluorescence signal was still observed in the CFn experimental group, indicating that the blockade is not complete.

[0141] 3. Bleed and sacrifice 6- to 8-week-old C57BL / 6 mice (purchased from Hunan Slack), soak in 75% ethanol (v / v) for 15 minutes, remove the spleen with ophthalmic scissors, place it in a sterile culture dish containing pre-cooled PBS, grind the spleen with a 10-mL syringe, filter the grinding solution through a 200-mesh nylon mesh into a 15-mL centrifuge tube, and centrifuge at 450 g for 5 minutes to collect the cells. Resuspend with 2 mL of red blood cell lysate (Sangon Biotech), lyse at 4 °C for 10 minutes. Add 10 mL of PBS to terminate the lysis, centrifuge at 450 g for 5 minutes to collect the cells. Resuspend with an appropriate amount of PBS and filter again through a 200-mesh nylon mesh. Count the cells after staining with 0.4% trypan blue. Transfer the lymphocytes isolated from the spleen to a 15-mL centrifuge tube, centrifuge at 450 g for 5 minutes, and discard the supernatant. Resuspend with MACS buffer (add 0.5% (v / v) FBS and 2 mM EDTA to 1×PBS) to make the cell density 1×108 cells / mL, and add cell sorting magnetic beads CD8a (Ly-2) MicroBeads (Miltenyi Biotec), mix well, and incubate at 4°C for 15 minutes. Fill up with MACS buffer, centrifuge at 450 g for 5 minutes, discard the supernatant to remove unlabeled magnetic beads. Resuspend the cells with an appropriate amount of MACS buffer to make the cell density 2×10 8 cells / mL. Place the sorting column on the magnetic cell sorter, place a 200-mesh nylon mesh on the sorting column, and rinse with MACS buffer. Filter the resuspended cells through the nylon mesh into the sorting column. After the liquid has drained, add 500 μL of MACS buffer to wash the sorting column, and repeat 3 times. Add 1 mL of MACS buffer to the sorting column, then remove the sorting column from the sorter and place it in a 15-mL centrifuge tube. Use the accompanying plunger to quickly push out the liquid in the sorting column to elute the T cells bound to the column, and stain and count with 0.4% trypan blue. Rinse the 24-well plate with sterile PBS 2 - 3 times. Add 200 μL of CD3 antibody (Abcam) dilution (4 μg / mL) to each well, cover the plate, seal with parafilm, and incubate at 4°C overnight or 37°C for 2 hours. Aspirate the antibody and wash 2 - 3 times with sterile PBS. Resuspend the T cells sorted by magnetic beads with RPMI-1640 complete medium, adjust the density to 1×10 6 cells / mL, and add CD28 antibody (Abcam) (2 μg / mL). Add the cell suspension to the pre-coated 25-well plate (500 μL / well), and culture in a 37°C CO2 incubator for 3 - 4 days. Then label with the fluorescent dye CFSE to obtain CFSE-labeled T cells. Stimulate MC38 cells with 50 ng / mL IFN-γ for 24 hours in advance to upregulate PD-L1 expression, then wash once with PBS to remove the medium, add trypsin (Thermo Fisher Scientific) and digest for 2 - 3 minutes until the cells detach, then add an equal volume of medium to terminate the digestion, collect the cell suspension, centrifuge at 100 g for 3 minutes, discard the supernatant, resuspend the precipitate with DMEM medium, and then inoculate at a density of 5×10 3 cells / well in a 96-well plate, and culture at 37°C for 4 hours to allow the cells to adhere. Add 5×10 4 CFSE-labeled T cells to each well, and simultaneously add PBS, HFn, CLP002, CMFn, and CFn (equivalent to the amount of CLP002 at 0.1 mg / mL) and continue to culture for 48 hours. Set 5 replicates for each group. Detect with a flow cytometer, and the results are as Figure 14 shown.

[0142] 4. After the co - culture of the above - mentioned MC38 cells and CFSE - labeled T cells was completed, the culture medium was collected, centrifuged at 450 g for 5 minutes to separate the T cells, and then the supernatant was taken. The supernatant was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was placed at - 80 °C for storage for later use. According to the instructions of the ELISA kit, first add 100 μL of capture antibody diluent into a 96 - well plate, seal it with a sealing film, and incubate overnight at 4 °C. Wash each well 4 times with 300 μL of Wash buffer, invert the residual buffer on the absorbent paper and gently pat it dry. Subsequent washes are carried out according to this step. To block non - specific binding and reduce background, add 200 μL of 1×Assay dilent A (BioLegend) to each well, seal it with a sealing film, incubate at room temperature on a shaker for 1 hour, and wash 4 times with Wash buffer. Add 100 μL / well of the standard or sample to the corresponding wells, seal with a sealing film, incubate at room temperature for 2 hours, and wash 4 times with Wash buffer. Add 100 μL of detection antibody diluent to each well, seal with a sealing film, incubate at room temperature for 1 hour, and wash 4 times with Wash buffer. Add 100 μL of the diluted horseradish peroxidase - labeled specific antibody (BioLegend) to each well, seal with a sealing film, incubate at room temperature on a shaker for 30 minutes, and wash 5 times with Wash buffer. Add 100 μL of TMB (3,3',5,5' - tetramethylbenzidine) substrate solution, incubate at room temperature in the dark for 10 minutes. Add 100 μL of stop solution to each well, and measure the absorbance at 450 nm with an enzyme - linked immunosorbent assay (ELISA) reader within 15 minutes. The experiment was set up with three replicates. The results are as Figure 15 shown. The concentration of IFN - γ in the supernatant after HFn treatment was not significantly different from that of the PBS group. Compared with HFn and CFn, the concentration of IFN - γ in the culture medium after CMFn treatment increased significantly.

[0143] 5. After the co - culture of the above - mentioned MC38 cells and CFSE - labeled T cells was completed, the culture medium was aspirated, and the cells were washed twice with PBS. Then, fresh medium containing MTT (0.5 mg / mL) was added, and the cells were continued to be cultured at 37 °C for 4 hours. Carefully aspirate the culture medium, add 150 μL of dimethyl sulfoxide (DMSO) to each well, place it on a shaker and oscillate at low speed for 10 minutes to fully dissolve the formazan crystals, and measure the absorbance of each well at 570 nm with an enzyme - linked immunosorbent assay (ELISA) reader. The experiment was set up with three replicates. The results are as Figure 16 shown. The viability of MC38 cells after HFn treatment was not significantly different from that of the PBS group. Compared with HFn and CFn, the viability of MC38 cells after CMFn treatment decreased significantly. The above results indicate that CMFn can restore the activity of T cells, promote the secretion of IFN - γ, enhance the killing effect of T cells on tumor cells, and the effect is better than that of CFn.

[0144] Study on the in vivo antitumor activity of enzyme-responsive recombinant ferritin

[0145] 1. Male C57BL / 6 mice aged 6 - 8 weeks were selected to establish a subcutaneous tumor mouse model. After quarantine, the purchased mice were acclimatized in the animal house for 1 - 2 weeks. MC38 cells were resuspended with sterile PBS and adjusted to a concentration of 5×10 6 cells / mL and placed on ice for later use. Each mouse was intraperitoneally injected with sodium pentobarbital (40 mg / kg). After the mouse was anesthetized, the hair in the groin area of the mouse was shaved. After skin disinfection, 100 μL of tumor cell suspension (tumor cell inoculation amount was 5×10 5 cells / mouse) was subcutaneously injected. The inoculated mice were placed on a thermostatic heating pad and waited for them to wake up naturally. When the tumor volume grew to 100 mm 3 , the tumor-bearing mice were randomly divided into 5 groups (5 mice / group), namely: (1) PBS group: injected with an equal volume of PBS as a control; (2) HFn group: intravenously injected with HFn (40 mg / kg) via the tail every four days; (3) CLP002 group: intraperitoneally injected with free CLP002 polypeptide (2 mg / kg) every two days; (4) αPD-L1 group: intraperitoneally injected with PD-L1 monoclonal antibody (Bio X Cell) (10 mg / kg) every four days; (5) CMFn group: intravenously injected with CMFn (40 mg / kg) via the tail every four days. During the treatment process, the body weight of the mice and the size of the tumors were measured every two days, and the tumor volume was calculated. On the 16th day after the start of treatment, the mice were sacrificed, the tumors were dissected, weighed and photographed. The changes in the tumor volume of the mice during the treatment process were as Figure 17 shown. Compared with the PBS group, injecting HFn had no obvious effect on tumor growth, and the tumors in both groups grew rapidly after the start of treatment. However, the change trend of the tumor volume was gentle after receiving CMFn treatment, indicating that tumor growth was inhibited. The inhibitory effect of CMFn on tumors was better than that of free CLP002 polypeptide and comparable to that of the αPD-L1 positive control group. After the treatment was completed, the mice were sacrificed and the tumors were dissected, weighed and photographed. As Figure 18 shown, there was no obvious difference in the tumor weight and size between the HFn group and the PBS group. Compared with the HFn group and the CLP002 group, the tumor weight and size in the CMFn group were significantly reduced. In addition, there was no obvious difference in the tumor weight between the CMFn group and the αPD-L1 group. As Figure 19 shown, there was no obvious difference in the body weight of the mice among the groups during the treatment process, and there was no sharp change in body weight, indicating that the drug treatment had no obvious effect on the growth of the mice.

[0146] 2. After bleeding the mice by enucleation after the above drug administration treatment, cut open the carotid artery to make the bleeding more sufficient, and surgically dissect the tumor. Cut the tumor tissue into small pieces as much as possible in a 5 mL centrifuge tube, and add 4 mL of collagenase VI (Sangon Biotech) (1 mg / mL) digestion solution to each tube. Seal the centrifuge tube with a sealing film and place it in a 37 °C constant temperature shaker for digestion for 1 hour at a rotation speed of 180 rpm. Filter through a 200-mesh nylon net into a 15 mL centrifuge tube, centrifuge at 450 g for 5 minutes, and discard the supernatant. Resuspend the precipitate with 6 mL of 40% (v / v) Percoll solution (Cytiva), and set the acceleration and deceleration of the centrifuge to 6 and 2800 g respectively and centrifuge for 20 minutes. Discard the supernatant, add 2 mL of red blood cell lysis solution to resuspend the precipitate, and lyse at 4 °C for 10 minutes. Fill up with 1×PBS, centrifuge at 800 g for 5 minutes to collect the cells. Resuspend with an appropriate amount of PBS, filter through a 200-mesh nylon net into a new 15 mL centrifuge tube, and count the cells after 0.4% trypan blue staining. Collect the cells in a 1.5 mL centrifuge tube (1×10 6 cells / tube), centrifuge at 450 g for 5 minutes to collect the cells, resuspend the cells with 50 μL of CD16 / 32 antibody (BioLegend) diluent (10 μg / mL), and block at 4 °C for 20 minutes. Add 50 μL of antibody diluent, incubate at 4 °C in the dark for 30 minutes. Fill up with PBS, centrifuge at 3000 g for 2 minutes, and discard the supernatant. Resuspend the cells with 300 μL of PBS to make the cell density 3 - 5×10 6 cells / mL. Pass through a 200-mesh filter into a flow tube and detect using a flow cytometer. The experiment was set up with three replicates. The proportions of various immune cells in the tumor tissue to the total immune cells are as Figure 20 shown. There were no significant differences in the proportions of various immune cells between the HFn group and the PBS group, indicating that unmodified HFn had no obvious effect on the tumor immune microenvironment. Compared with the HFn group, the proportions of CD4+ T cells, CD8+ T cells, NK cells, and NKT cells in the CD45+ cells in the tumor tissue of mice after CMFn treatment were significantly increased, and the changes in these immune cell populations were similar to those of the positive control group αPD-L1. The above results indicate that CMFn can effectively promote the anti-tumor immune response of mice and achieve a therapeutic effect similar to that of monoclonal antibodies. In addition, compared with injecting free CLP002 polypeptide, CMFn induced a stronger anti-tumor immune response, indicating that the loading of ferritin enhanced the immunotherapeutic effect of CLP002 polypeptide.

[0147] Example 4 Preparation and Characterization of Enzyme-Responsive Functionalized Ferritin

[0148] 1. Encapsulation of oxaliplatin (OXA) by pH-mediated depolymerization and reassembly. HFn or CMFn was added to 50 mM Tris-HCl buffer (pH 2.5) with a final protein concentration of 2 μM, and stirred on ice for 5 minutes. Then, oxaliplatin with a final concentration of 4 mM was added and mixed well. The pH of the reaction system was adjusted to 4.0 by slowly dropping 50 mM Tris-HCl buffer (pH 9.2). Under the condition of pH 4.0, the denatured ferritin began to reassemble. The reaction system was transferred to a dialysis bag (molecular weight cut-off of 1 kD) and dialyzed overnight at 4 °C in 50 mM Tris-HCl buffer (pH 7.4) to allow the complete self-assembly of ferritin, while encapsulating oxaliplatin in the inner cavity of the nanocage. After dialysis, it was centrifuged at 12000 rpm for 10 minutes to remove possible precipitates, and then dialyzed in PBS for 24 hours to remove unencapsulated oxaliplatin, obtaining HFn@OXA and CMFn@OXA. The morphological changes of CMFn during the encapsulation process were observed by transmission electron microscopy. As Figure 21 shown, when HFn and CMFn were in the buffer at pH 2.5, the particle size increased significantly, probably due to the depolymerization of ferritin subunits resulting in an increase in volume. After the pH was adjusted back to 7.4, the ferritin subunits reassembled self-assembled, and oxaliplatin was encapsulated inside the protein cage. Transmission electron microscopy images showed that the generated HFn@OXA and CMFn@OXA were uniformly sized spheres, consistent with the morphology before drug encapsulation.

[0149] 2. Ultraviolet-visible spectroscopy detection of oxaliplatin (OXA) using an ultraviolet spectrophotometer. After turning on the instrument, preheat it for 20 minutes, set the software, set the detection wavelength range to 200 - 800 nm, and the detection step to 1 nm. First, load PBS into the cuvette and detect the baseline. Then load the sample into the cuvette and detect the ultraviolet-visible spectrum of the sample. As Figure 22 shown, we first detected the ultraviolet-visible spectra of oxaliplatin at different concentrations (15.6, 31.3, 62.5, 125, 250, 500 μg / mL). The results showed that the maximum absorption wavelength of oxaliplatin was at 251 nm. We plotted the standard curve of oxaliplatin by detecting the absorbance at 251 nm. In addition, we compared and analyzed the ultraviolet-visible spectra of different concentrations of OXA, CMFn, and CMFn@OXA. As Figure 23 shown, the ultraviolet absorption of OXA was detected in CMFn@OXA, indicating that OXA was successfully encapsulated.

[0150] 3. Load HFn@OXA or CMFn@OXA (10 mg / mL) into a dialysis bag with a molecular weight cut-off of 10 kDa and seal it with a clip. In 50 mL centrifuge tubes, respectively load 30 mL of PBS at pH 7.4 and pH 5.0 to simulate the normal physiological environment and the lysosomal environment, respectively. Then place the dialysis bag containing the sample into the centrifuge tube and wrap it with aluminum foil to avoid light. Place the centrifuge tube vertically in a thermostatic shaking incubator, set the temperature to 37 °C and the rotation speed to 100 rpm. At 0, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 hours respectively, pipette 3 mL of PBS from the tube and supplement it with the same volume and pH of PBS to ensure that the total volume of the release medium remains unchanged. Measure the drug concentration in the PBS by HPLC and calculate the percentage of drug release. The experiment is set up with three replicates. The results are as Figure 24 shown. CMFn@OXA showed a drug release trend similar to that of HFn@OXA. Under the condition of pH 7.4, the release of oxaliplatin from CMFn@OXA was relatively slow, with 11.8% released after 48 hours of incubation. While under the condition of pH 5.0, oxaliplatin was rapidly released and reached 95.7% release after 48 hours.

[0151] 4. Load CMFn@OXA (10 mg / mL) into a dialysis bag with a molecular weight cut-off of 10 kDa and seal it with a clip. In 50 mL centrifuge tubes, respectively load 30 mL of PBS and 10% (v / v) FBS, where 10% (v / v) FBS is used to simulate the plasma environment. Then place the dialysis bag containing the sample into the centrifuge tube and wrap it with aluminum foil to avoid light. Place the centrifuge tube vertically in a thermostatic shaking incubator, set the temperature to 37 °C and the rotation speed to 100 rpm. At 0, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, and 72 hours respectively, pipette 3 mL of PBS or FBS from the tube and supplement it with the same volume of fresh release medium to ensure that the total volume of the release medium remains unchanged. Measure the drug concentration in the PBS by HPLC and calculate the percentage of drug release. The results are as Figure 25 shown. After incubating in FBS for 72 hours, only 12.5% of the oxaliplatin in CMFn@OXA was released, showing no significant difference from that in PBS. The above results indicate that CMFn@OXA has strong stability in the physiological environment of blood and the encapsulated oxaliplatin is not easily leaked.

[0152] 5. Use the CCK-8 kit to detect the cytotoxicity of the drug. When MC38 cells, HCT116 cells, and HUVEC cells grow to the logarithmic growth phase, digest and collect the cells for cell counting. After diluting the cells to an appropriate concentration, inoculate them into 96-well plates (5×10 3(number / hole), add the same volume of culture medium to the blank holes, and add PBS around the 96-well plate for moisturizing. Place the 96-well plate in an incubator at 37°C and culture overnight. Then add OXA, HFn@OXA or CMFn@OXA (calculated as OXA, with concentrations of 0, 0.156, 0.313, 0.625, 1.250, 2.500 μg / mL respectively) and treat for 72 hours, with 5 replicates for each concentration. After the drug treatment, add 10 μL of CCK-8 solution to each well, gently shake the culture plate several times, and then continue to incubate in the cell incubator for 1 - 4 hours. Measure the absorbance at 450 nm with a microplate reader and calculate the cell viability. The results are as Figure 26 shown. For HCT116 and MC38, CMFn treatment did not show obvious cytotoxicity, while both CMFn@OXA and OXA showed concentration-dependent cell killing. Notably, at the same drug concentration, the cytotoxicity of CMFn@OXA to HUVEC was less than that of free OXA, indicating that encapsulation by CMFn reduced the toxicity of OXA to normal cells.

[0153] 6. Dissolve CMFn@OXA in PBS solution and adjust the pH to 8.5 with 1 M NaHCO3 solution. Prepare sulfonated Cy5.5 NHS ester (CAS No.: 2419286-92-1) in DMSO at a concentration of 10 mg / mL, and then slowly add it dropwise to the CMFn@OXA solution to make the molar ratio of Cy5.5 to CMFn 1:10. Stir slowly in the dark at room temperature for 45 minutes. Place the mixed solution in PBS and dialyze in the dark at 4°C for 24 hours, changing PBS every 6 hours to remove free Cy5.5 molecules. Filter the dialyzed solution through a 0.22 μM syringe filter to obtain Cy5.5-CMFn@OXA. Use a BCA protein concentration detection kit to detect the concentration of CMFn, use an ultraviolet spectrophotometer to measure the absorbance at 650 nm to detect the concentration of Cy5.5, and calculate the fluorescence labeling efficiency F / P (the molar ratio of fluorescein to protein). Seed MC38 cells into confocal dishes, culture at 37°C for 12 hours to allow the cells to adhere, then add Cy5.5-CMFn@OXA. After incubating for 0.5, 1, 4, 12, 24 hours respectively, aspirate the culture medium, wash once with PBS, add the culture medium containing Hoechst 33342 nuclear stain (5 μg / mL), incubate at 37°C for 20 minutes, aspirate the culture medium, wash three times with PBS, and observe using a confocal laser scanning microscopy (CLSM). The experiment was set up with three replicates. As Figure 27As shown, the fluorescence signal of Cy5.5-CMFn@OXA could be observed in cells after incubation for 0.5 h, and the signal gradually increased with the prolongation of time, indicating that Cy5.5-CMFn@OXA was continuously taken up by cells and accumulated in cells.

[0154] 7. After incubating Cy5.5-CMFn@OXA with MC38 cells for 12 h, they were stained with Hoechst 33342 and LysoSensor TM Green DND-189 lysosome probe (2 μM), and then observed by CLSM. The experiment was set up with three replicates. As Figure 28 shown, the fluorescence signal of Cy5.5-CMFn@OXA co-localized with LysoSensor, indicating that CMFn@OXA was transported to lysosomes after being taken up by cells.

[0155] 8. An MC38 murine colon cancer model was constructed (the same method as in Example 3). When the tumor grew to about 100 mm 3 in size, the tumor-bearing mice were randomly divided into 5 groups (5 mice / group), and 200 μL of PBS, Cy5.5-HFn@OXA (the preparation method was the same as that of the above Cy5.5-CMFn@OXA, just replace CMFn@OXA with HFn@OXA), or Cy5.5-CMFn@OXA (Cy5.5 concentration was 0.5 mg / mL) were injected via the tail vein respectively. At 4, 8, 12, and 24 h after injection, imaging was performed using a Bruker In Vivo Xtreme II small animal in vivo imaging system, and the excitation wavelength and emission wavelength were set at 630 nm and 700 nm respectively to observe the distribution of the drug in vivo over time. As Figure 29 shown, no fluorescence signal was observed in the PBS control group at the 4 time points, while strong fluorescence signal appeared at the tumor site 4 h after injection of Cy5.5-CMFn@OXA, indicating its extensive enrichment in the tumor, and this enrichment continued until 24 h. In contrast, 4 h after injection of Cy5.5-HFn@OXA, although fluorescence signal could also be observed at the tumor site, it was weaker compared to the lungs, and after 24 h, the fluorescence signal was mainly distributed at the tumor site and the lungs. The mice were sacrificed and dissected 24 h after injection, and the tumors, hearts, livers, spleens, lungs, and kidneys of the mice were isolated for fluorescence imaging. As Figure 30 shown, the strongest fluorescence signal was found in the tumors of the Cy5.5-CMFn@OXA group, while relatively strong fluorescence signal also appeared in the lungs of the Cy5.5-HFn@OXA group.

[0156] Example 5 In Vivo Antitumor and Biosafety Studies of Enzyme-Responsive Functionalized Ferritin

[0157] 1. The construction of the MC38 murine colon cancer model was as described in Example 3. When the tumor grew to 100 mm 3 , the tumor-bearing mice were randomly divided into 5 groups (5 mice / group), namely: (1) PBS group: an equal volume of PBS was injected via the tail vein as a control; (2) Free OXA group: free oxaliplatin (1 mg / kg) was injected via the tail vein; (3) HFn@OXA group: HFn@OXA (1 mg / kg oxaliplatin equivalent) was injected via the tail vein; (4) αPD-L1+OXA group: the anti-PD-L1 monoclonal antibody (5 mg / kg, intraperitoneal injection) was used in combination with oxaliplatin (1 mg / kg, tail vein injection); (5) CMFn@OXA group: CMFn@OXA (1 mg / kg oxaliplatin equivalent) was injected via the tail vein. Administration was performed once every four days after the start of treatment. During the treatment process, the body weight and tumor volume of the mice were measured every two days. After the treatment was completed, the mice were sacrificed, and the tumor tissues were dissected for weighing, photographing, and subsequent processing. The change in the tumor volume of the mice over time is as shown in Figure 31 . In the PBS control group, the tumor volume increased rapidly after the start of treatment. The change trend of the tumor volume in the CMFn@OXA administration group was the gentlest. On the 18th day of treatment, the tumor volume was 18.8% of that in the PBS control group, and the effect was better than that in the αPD-L1+OXA administration group (45%). In addition, the growth rate of the tumors in the HFn@OXA group was slower than that in the Free OXA group, indicating that encapsulation with ferritin could effectively enhance the anti-tumor effect of OXA. After the treatment was completed, the tumors of the mice were dissected for weighing and photographing, as shown in Figure 32 . After CMFn@OXA treatment, the volume and size of the tumors were significantly smaller than those in other experimental groups, which was consistent with the previous conclusion. In addition, as shown in Figure 33 , there were no drastic changes in the body weight of the mice during the treatment process. We also statistically analyzed the survival rate of the mice in each group. Figure 34 is the survival curve of the mice. The median survival time of the PBS group was 25 days, and the median survival time of the tumor-bearing mice after CMFn@OXA treatment was extended to 49 days.

[0158] 2. After the treatment was completed, blood was collected from the eyes of all mice for serum biochemical analysis. The mice were grasped, and the skin around the eyes was compressed with the thumb and index finger to make the eyeballs protrude and become congested. The eyeballs were quickly removed with an ophthalmic forceps, and the mice were held head down to allow the blood to drip into a centrifuge tube by gravity. The mouse blood samples were placed at room temperature for 30 minutes for coagulation, and then centrifuged at 1500 g for 10 minutes at room temperature. The upper-layer serum was carefully aspirated using a pipette for serum biochemical analysis. The biochemical analysis indicators included aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea (UREA), creatinine (CREA), lactate dehydrogenase (LDH), and creatine kinase (CK). The results are as shown in Figure 38As shown, no abnormalities were found in the six indicators after treatment with CMFn@OXA.

[0159] 3. Place the above-stripped tumor tissue blocks in pre-cooled PBS to wash away the residual blood. Transfer the tissue blocks to a 5 mL centrifuge tube, add pre-cooled PBS (add 5 mL PBS per 1 g of sample), and then break them up with a homogenizer. The resulting homogenate is further disrupted by ultrasound, and the temperature is lowered by ice bath during the ultrasound process. The prepared homogenate is centrifuged at 12,000 rpm for 10 minutes, and the supernatant is taken to measure the total protein concentration. The experiment is set up with three replicates. The results are as Figure 35 shown. Compared with the PBS group, the levels of IFN-γ and TNF-α in the tumor tissue did not change significantly after injection of free OXA, while the cytokine levels increased significantly after injection of HFn@OXA. Compared with the OXA group and HFn@OXA, the levels of IFN-γ and TNF-α in the tumor increased significantly after injection of CMFn@OXA, and there was no significant difference from the αPD-L1+OXA combination treatment group.

[0160] 4. After the treatment is over, the mice are sacrificed and dissected, and the tumors, hearts, livers, spleens, lungs, and kidneys are isolated. Use a sharp knife to cut some tissue blocks, with the thickness of each block not exceeding 0.5 cm, and soak them in 4% paraformaldehyde for fixation for 24 to 48 hours. Subsequently, use alcohol with gradually increasing concentrations as a dehydrating agent to gradually remove the water in the tissue blocks. Next, soak the tissue blocks in xylene to make them transparent, and replace the alcohol in the tissue blocks with xylene. Put the transparently treated tissue blocks into melted paraffin, and then place them in a constant-temperature air blast drying oven for insulation. After the paraffin has completely penetrated into the tissue blocks, quickly pick up the tissue blocks that have been soaked with paraffin and put them into melted paraffin for embedding until the paraffin cools and solidifies. Fix the embedded wax blocks on a microtome and cut them into thin slices with a thickness of 5 to 8 microns. Subsequently, place the cut thin slices in hot water to flatten them, then stick them onto glass slides, and place them in an incubator at 45°C for drying.

[0161] 5. Send the above-prepared tumor sections to Hangzhou Zhiqi Biotechnology Co., Ltd. for immunohistochemistry experiments. The results are as Figure 36 shown. No fluorescent signals of T cells were detected in the tumor tissue of the PBS group. The infiltration of tumor-infiltrating T cells increased slightly after injection of OXA and HFn@OXA, while the infiltration of T cells in the tumor tissue increased significantly after injection of CMFn@OXA. The above results indicate that CMFn@OXA can effectively promote the immune infiltration of T cells and enhance the anti-tumor immune response.

[0162] 6. Hematoxylin (H) is a basic dye that can stain cell nuclei and ribosomes in cells blue-violet; while Eosin (E) is an acidic dye that can stain cytoplasm red or light red. Before staining, the prepared heart, liver, spleen, lung, and kidney sections were baked at 60 °C for 2 hours, and the paraffin in the sections was removed with xylene by soaking 3 times, 15 minutes each time. Subsequently, they were successively soaked in absolute ethanol or ethanol solutions of 100%, 95%, 90%, 80%, 70% (v / v) for 5 minutes each, and finally placed in tap water. Then, the sections were soaked in hematoxylin aqueous solution for 15 minutes to stain the cell nuclei, and then rinsed with tap water for 3 to 5 minutes. Subsequently, they were differentiated with 0.5% hydrochloric acid ethanol for 5 to 30 seconds and rinsed with tap water for 1 to 3 minutes. Next, they were blued with 0.2% (v / v) ammonia water for 30 to 60 seconds and rinsed with tap water again for 5 to 10 minutes. Finally, the fully hydrated sections were soaked in eosin staining solution for 5 to 15 minutes to stain the cytoplasm. Subsequently, gradient ethanol was used for dehydration, and then xylene was used for clarification. After the sections were air-dried, they were sealed with neutral gum. The results are as Figure 37 shown that no tissue damage to major organs was observed after CMFn@OXA administration.

[0163] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An enzyme-responsive recombinant ferritin with PD-L1 blocking function, characterized in that: The amino acid sequence is as shown in SEQ ID NO.

1.

2. The coding gene of the enzyme-responsive recombinant ferritin according to claim 1, characterized in that: The nucleotide sequence is as shown in SEQ ID NO.

2.

3. The preparation method of the enzyme-responsive recombinant ferritin with PD-L1 blocking function according to claim 1, characterized in that, It includes the following steps: (1) The coding gene sequence of the recombinant ferritin as shown in SEQ ID NO.2 and the pET-MBP-HRV 3C expression plasmid are respectively double digested with BamHⅠ and HindⅢ enzymes, and then ligated to obtain the CMFn expression plasmid; (2) The CMFn expression plasmid is transformed into Escherichia coli competent cells, and after induced expression and purification, the enzyme-responsive recombinant ferritin with PD-L1 blocking function is obtained; The conditions for the induced expression in step (2) are: 0.5 mM isopropyl-β-D-thiogalactopyranoside, cultured at 37 ± 1 °C for 6 - 10 hours; The purification in step (2) is carried out through a Ni-IDA affinity chromatography column, and the eluent used is a 300 mmol / L imidazole solution.

4. Use of the enzyme-responsive recombinant ferritin with PD-L1 blocking function as claimed in claim 1 in the preparation of a PD-1 blocker, an anti-tumor drug carrier, and / or an anti-tumor drug.

5. Use of the combination of the enzyme-responsive recombinant ferritin with PD-L1 blocking function as claimed in claim 1 and a chemotherapeutic drug in the preparation of an anti-tumor drug.

6. The application according to claim 5, characterized in that: The chemotherapeutic drug is oxaliplatin.

7. An enzyme-responsive engineered ferritin nanoparticle, characterized in that: It is obtained by encapsulating the chemotherapeutic drug with the enzyme-responsive recombinant ferritin with PD-L1 blocking function as claimed in claim 1; the chemotherapeutic drug is oxaliplatin.

8. The preparation method of the enzyme-responsive engineered ferritin nanoparticles according to claim 7, characterized in that, It includes the following steps: The enzyme-responsive recombinant ferritin with PD-L1 blocking function as claimed in claim 1 is added to a buffer solution with a pH of 2.5 ± 0.1, stirred on ice, then the chemotherapeutic drug is added, and the pH is adjusted to 4.0 ± 0.1 to make the denatured ferritin start to reassemble. Then the reaction system is transferred to a dialysis bag and dialyzed in a buffer solution with a pH of 7.4 at 4 °C to make the ferritin completely self-assemble, and at the same time encapsulate oxaliplatin in the inner cavity of the nanocage to obtain the enzyme-responsive engineered ferritin nanoparticles; The buffer solution with a pH of 2.5 ± 0.1 and the buffer solution with a pH of 7.4 are both 50 mmol / L Tris-HCl buffer solutions; The concentration of the protein in the reaction system is 2 μmol / L, and the concentration of the chemotherapeutic drug is 4 mmol / L; The cut-off molecular weight of the dialysis bag is 1 kD; The dialysis time is 10 - 12 hours.

9. Use of the enzyme-responsive engineered ferritin nanoparticles as claimed in claim 7 in the preparation of a PD-1 blocker and / or an anti-tumor drug.

10. The application according to any one of claims 4 to 6 and 9, characterized in that: The tumor is a malignant tumor including colon cancer, ovarian cancer, breast cancer, gastric cancer, pancreatic cancer, non-small cell lung cancer, melanoma, testicular tumor, lymphoma, and head and neck tumor.

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