Tumor microenvironment response fusion protein-metal ion compound prodrug system as well as preparation method and application thereof

Abstract

The invention provides a novel platform for tumor immunotherapy. The platform is formed by self-assembly of full-heavy-chain human ferritin which can be jointly fused and expressed by a polypeptide prodrug mask which can be activated by protease and a mitochondrial disrupting peptide, and can specifically target a tumor microenvironment and induce ICD death of tumors. Meanwhile, an ion channel is formed in a protein shell of the platform, so that immunocompetence metal ions can be loaded in a cavity of the platform, and time-space synchronous anti-tumor immune activation can be carried out in cooperation with ICD. As the mitochondrial disrupting peptide and the metal ions can be optimized according to tumor types, the platform can be used for treating various tumors.

Description

Technical Field

[0001] The invention relates to a tumor microenvironment-responsive fusion protein-metal ion complex prodrug system and a preparation method and application thereof. Background Art

[0002] Immunogenic cell death (ICD) is a mechanism of action that can generate antitumor immune responses. ICD promotes the expression of calreticulin (CRT) as an “eat me” signal and releases damage-associated molecular patterns (DAMPs) and tumor-associated antigens, which can serve as in situ tumor vaccines. Most traditional anticancer drugs used to induce ICD do so by triggering “side branch effects”, resulting in limited antitumor immune responses. To effectively induce ICD, the generation of sustained reactive oxygen species (ROS) leading to endoplasmic reticulum (ER) stress is essential. Given that mitochondria are the major organelles producing ROS in eukaryotic cells, direct manipulation of mitochondria to enhance ROS production may be a more direct and effective approach. Bioactive peptides, such as naturally toxic peptides, borane mimetic peptides, and cationic amphipathic peptides, can induce mitochondrial membrane rupture, leading to the accumulation of ROS and ultimately cell death. Therefore, screening for ICD inducers from these mitochondrial disrupting peptides may be a promising biopharmaceutical strategy for generating in situ tumor vaccines.

[0003] At the same time, there are challenges in the spatiotemporal synchronization of tumor-specific immune responses after stereotactic induction of ICD. The key role of metal ions, such as aluminum, magnesium, zinc, and manganese, in regulating innate immunity is increasingly recognized. Therefore, metal ions may be considered as potential enhancers of tumor-specific immune responses. However, highly water-soluble metal ions are difficult to properly load into traditional delivery systems, resulting in low loading efficiency and premature leakage in the circulation. In addition, under physiological conditions, PO 3- The presence of metal ions in abundance may lead to the formation of phosphate nanoparticles when they leak out, causing unexpected adverse reactions. Therefore, appropriate carriers are needed to improve the targeting specificity and bioavailability of metal-based drugs.

[0004] Ferritin (Fn) is a widely existing iron storage protein, composed of 24 subunits, which aggregate to form a cage structure with an outer diameter of 12nm and an inner cavity of 8nm. The cage structure of the ferritin can encapsulate metal ions such as manganese, magnesium, copper, zinc, iron, aluminum, potassium, sodium, etc. More importantly, CD71, as a receptor for Fn, is overexpressed in most human tumors, allowing Fn to be preferentially internalized by tumor cells. Due to its reversible cage structure and natural metal encapsulation properties, as well as its thermal stability, biocompatibility and non-immunogenicity, Fn has become an ideal tumor-targeted delivery platform for metal-based therapy, but Fn still has the following problems. First, CD71 is significantly expressed in the liver, resulting in Fn being preferentially intercepted by the liver instead of targeting tumors. Secondly, the stability of the Fn-drug complex is not ideal, and more than 30% of drug leakage was observed within the first week. In addition, the plasma half-life of Fn is relatively short (about 2 hours).

[0005] Therefore, the art desires a preparation or complex that can overcome the defects of Fn and simultaneously achieve the spatiotemporal synchronous activation of tumor-specific immune response after stereotactic induction of ICD. Summary of the invention

[0006] Purpose of the Invention

[0007] The purpose of the present invention is to provide a tumor microenvironment-responsive fusion protein-metal ion complex prodrug system to overcome the defects of the prior art.

[0008] Technical Solution

[0009] A tumor microenvironment-responsive fusion protein-metal ion complex prodrug system, characterized in that the system comprises a tumor-targeted ICD-inducing drug carrier and an immune-activating metal ion content.

[0010] The system is characterized in that the tumor-targeted ICD-inducing drug carrier is a human heavy chain ferritin derivative polymerized to form nanoparticles with cavities, and the metal ions are loaded in the cavities.

[0011] The system is characterized in that the human heavy chain ferritin derivative spontaneously forms a cage-like 24-mer to encapsulate metal ions.

[0012] The system is characterized in that the human heavy chain ferritin derivative comprises a PSTAG shielding sequence, a tumor microenvironment enzyme response sequence, an ICD inducing peptide, an intracellular release sequence and an intraluminal modified human heavy chain ferritin; and the metal ions include manganese, magnesium, copper, zinc, iron, aluminum, potassium or sodium.

[0013] The PSTAG shielding sequence is:

[0014] GSGSSGPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPAG, see SEQ ID NO.1,

[0015] or

[0016] GSGSSGPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGP SATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTG, see SEQ ID NO.2;

[0017] or

[0018] GSGSSGPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSATPGSTGPTSAPGSTGPSATPGTSGPSGTSAPTGPSGTPASTGATPSGTSGPSATPGTSGPSGTPSPTAGPSATPGSTGPTSAPGSTGPSTGSPATGPST, see SEQ ID NO.3;

[0019] The tumor microenvironment enzyme response sequence is PRHLRNLVPMVATV, see SEQ ID NO.4;

[0020] The ICD inducing peptide is GQVGRQLAIIGDDINR, see SEQ ID NO.5; CGTNVFNATFHIWHSGQFGT, see SEQ ID NO.6; AVPIAQK, see SEQ ID NO.7;

[0021] GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE, see SEQ ID NO.8;

[0022] KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL, see SEQ ID NO.9;

[0023] PAWRKAFRWAWRMLKKAA, see SEQ ID NO.10;

[0024] KLAKLAKKLAKLAK, see SEQ ID NO. 11;

[0025] KLNFRQKLLNLISKLFCSGT, see SEQ ID NO. 12;

[0026] The intracellular release sequence is SLVR, see SEQ ID NO.13;

[0027] The amino acid sequence of the lumen-modified human heavy chain ferritin is

[0028] MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNESHHHHHHHH, see SEQ ID NO.14.

[0029] The system is characterized in that the amino acid sequence of the human heavy chain ferritin derivative is:

[0030] MGKLAKLAKKLAKLAKGGSLVRGGMTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIK KPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNESHHHHHHHH, see SEQID NO.15、

[0031] or

[0032] MGGSGSSGPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPAGEFPRHLRNLVPMVATVGGKLAKLAKKLAKLAKGGSLVRGGMTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNESHHHHHHHH, see SEQ ID NO. 16,

[0033] or

[0034] MGGSGSSGPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTGEFPRHLRNLVPMVATVGGKLAKLAKKLAKLAKGGSLVRGGMTTASTSQVRQNYHQDSEAAINRQINLELYAS YVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNESHHHHHHHH, see SEQ ID NO. 17,

[0035] or

[0036] MGGSGSSGPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTGEFPRHLRNLVPMVATVGGKLAKLAKKLAKLAKGGSLVRGGMTTASTSQVRQNYHQDSEAAINR QINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNESHHHHHHHH, see SEQ ID NO.18.

[0037] The preparation method of the system is characterized by comprising the following steps: fusing and expressing an ICD inducing peptide on the outside of ferritin; utilizing the ion channel inherent in the ferritin structure, opening the ferritin ion channel and the PSTAG winding shield by heating, adding metal ions thereto to allow them to enter the ferritin, restoring to room temperature so that the ferritin ion channel is closed and the PSTAG restores the spatial shielding, thereby obtaining a fusion protein-metal ion complex whose inner cavity contains metal ions.

[0038] The system is used in preparing drugs for preventing or treating tumors.

[0039] The application is characterized in that the tumor is breast cancer, colorectal cancer, lung cancer, ovarian cancer, melanoma, gastric cancer, pancreatic cancer, bladder cancer, kidney cancer, prostate cancer or brain cancer. The drug loading of the prodrug system is 900-3200 metal ions per cage-shaped 24-mer.

[0040] Preferably, the PSTAG shielding sequence, the tumor microenvironment enzyme response sequence, the ICD induction sequence and the intracellular release sequence are coupled via a GG or EF linker sequence;

[0041] The present invention also relates to a method for preparing the fusion protein-metal ion complex prodrug system. Specifically, the method comprises the following steps:

[0042] Step (1), preparing and purifying the lumen-modified human heavy chain ferritin derivative

[0043] The full-length sequence of human H-ferritin was obtained using the NCBI database, and PSTAG, tumor microenvironment response site, and ICD induction peptide functional sequence were introduced into its N-terminus to design the protein sequence of the fusion protein. After codon optimization, the cDNA of the fusion protein was constructed into the expression vector pET-28a (GenScript); then, the fusion protein-pET-28a was transformed into the expression strain BL21 (DE3) (Beyotime Biotechnology) using the prokaryotic expression system, and the expression was induced by IPTG, and then the human HFn protein was purified;

[0044] The specific method of expression and purification is as follows: Induce protein expression with 0.5mM IPTG at 37°C for 8 hours. Collect the bacteria and resuspend them with 20mM Tris-HCl (pH 8.0) buffer. Obtain bacterial lysate by ultrasonic treatment, and then centrifuge (12,000g, 30min, 4°C). Collect the supernatant and heat it at 70°C for 15min, and centrifuge it again (12,000g, 30min, 4°C). Collect the supernatant and use Q-Sepharose FastFlow and superdex TM The purified protein was purified by 200 pg column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed using 12% (v / v) polyacrylamide gel. Then, the purest fraction was collected, ultrafiltered using a 100 kDa centrifugal ultrafiltration tube and the buffer was replaced with 20 mM HEPES to obtain a fusion protein concentrate.

[0045] Step (2), using the lumen-modified human heavy chain ferritin derivative to load manganese ions

[0046] The lumen-modified human heavy chain ferritin derivative was diluted with 20 mM HEPES pH 8.0 to a final concentration of 1.5 mg / mL in a final volume of 50 mL and gently stirred in a 60 °C constant temperature water bath for 30 min;

[0047] A manganese chloride solution (450 mM) prepared with deionized water was dropwise pumped into the reaction mixture at a rate of 130 μL / min (gently stirred in a 60 °C water bath) and stirred for 80 min;

[0048] The reaction mixture was gently stirred in a water bath at 60°C for 160 min. The reaction mixture was then cooled to room temperature naturally and centrifuged (8,000 g, 30 min, 4°C) to remove denatured proteins.

[0049] The supernatant was placed in a 50 kDa dialysis bag and dialyzed against HEPES buffer at 4°C overnight to remove free manganese ions, thereby obtaining a fusion protein-metal ion complex.

[0050] Step (3): Calculation and quantification of drug loading of fusion protein-metal ion complex

[0051] The fusion protein concentration was determined using a BCA protein assay kit, and the encapsulated manganese was quantitatively determined using ICP-OES 5110 to deduce the metal ion loading of the fusion protein-metal ion complex.

[0052] principle:

[0053] like Fig.10 As shown in a, the human heavy chain ferritin derivative is a fusion protein formed by coupling the PSTAG shielding sequence, the tumor microenvironment enzyme response sequence, the ICD induction sequence and the intracellular release sequence with the intraluminal modified human heavy chain ferritin; the following functions are realized: ① PSTAG shielding sequence: reduce the leakage of metal ions in the ferritin cage, prolong the half-life of the fusion protein, improve the stability of the fusion protein, improve the in vivo distribution of the fusion protein, reduce off-target liver enrichment, and reduce off-target toxicity. ② Tumor microenvironment response site: can be cut by enzymes highly expressed in the tumor microenvironment, remove the shielding sequence, and release the fusion protein that exerts anti-tumor effects. ③ ICD inducing peptide: a cell-killing peptide that can act on the mitochondria of tumor cells and induce ICD death of tumor cells. ④ Intracellular cleavage site: after ferritin carries the ICD inducing short peptide into the tumor cell, it can separate the ICD inducing short peptide from ferritin, so that the ICD inducing short peptide can act on the mitochondria and play the role of inducing ICD.

[0054] like Fig.10 As shown in Figure b, the fusion protein-metal ion complex prodrug system has a strong anti-tumor effect after intravenous injection, and its mechanism of action is: after the complex enters the body, MMP14 cleavage occurs in the tumor microenvironment, PSTAG is removed, KLA-Fn is exposed, and then CD71 mediates entry into the cell. The active molecule KLA is released after intracellular cleavage, acts on mitochondria, and induces increased ROS levels. Tumor cells then undergo ICD and STING pathway activation, thereby mediating DC maturation and T cell activation, and achieving a systemic immune response. In the present invention, the experimental results show that the fusion protein-metal ion complex can successfully induce ICD in tumor cells, and has good tumor targeting, while activating the activity of immune cells, it can effectively inhibit the occurrence and development of tumors.

[0055] Beneficial Effects

[0056] The present invention constructs a new fusion protein-metal ion complex prodrug system, which includes a tumor-targeted ICD-inducing drug carrier and an immune-activating metal ion content. The tumor-targeted ICD-inducing drug carrier is a nanoparticle with a cavity formed by polymerizing a human heavy chain ferritin derivative, and the metal ions and drugs are loaded in the cavity. The present invention has the following advantages:

[0057] 1. If Figure 7 As shown, the complex significantly reduces liver accumulation, overcoming the defect of the prior art that Fn is preferentially intercepted by the liver instead of targeting the tumor; Figure 6 The half-life is prolonged as shown Figure 7 As shown in the figure, it reaches tumor site enrichment in about 8 hours, which has better tumor targeting than the prototype ferritin control without environmental response. In addition, the fusion protein-metal ion complex is less enriched in the liver, which improves the safety of the system.

[0058] 2. If Figure 3 As shown, the leakage of the complex within 24 hours at 4° C. and 37° C. is less than 10%, and the leakage is very small, thus overcoming the technical defect of unsatisfactory stability of Fn-drug complex in the prior art.

[0059] 3. If Figure 2 As shown, the complex can successfully induce ICD in tumor cells in a MMP14-dependent manner, while causing little damage to healthy cells, indicating that the present invention has no toxic side effects and is safer.

[0060] 4. If Figure 5 As shown, the complex encapsulates metal ions (such as Na + 、Al 3+ 、Zn 2+ , Cu 2+ , K + , Ca 2+ , Fe 2+ , Fe 3+ Mg 2 + , Mn 2+ etc.) increase the activation of DC cells and CD8+T cells, inducing better immune response activation. Figure 8 As shown, the complex of the present invention itself can be used as a drug to prevent the occurrence of tumors. Fig. 9 It has been shown that it can also inhibit tumor growth.

[0061] 5. If Figure 4 As shown, the present invention can further be used as a carrier to encapsulate drugs (anti-tumor drugs, such as doxorubicin) to increase the anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Characterization of a series of lumen-modified human heavy chain ferritin derivatives and fusion protein-metal ion complexes. a shows the KLA-Fn, P 54 PKF, P 108 PKF, P 216b is the dynamic light scattering result of PKF; b is the transmission electron microscopy method for KLA-Fn, P 54 PKF, P 108 PKF, P 216 PKF is used for characterization; c is P 216 PKF@Mn 2+ Dynamic light scattering results of the complex; d is P 216 PKF@Mn 2+ Transmission electron microscopy characterization of the composites.

[0063] Figure 2 This is a study on the ICD induction activity of the fusion protein in vitro. a is P 216 Laser confocal scanning microscopy fluorescence images of L02, MCF-7, MDA-MB-231, and 4T-1 cells stained with JC-1 after PKF treatment for 24 h; b is P 216 Quantitative results of cells treated with PKF and stained with JC-1 by fluorescence microplate reader; c is P 216 After PKF treatment, cells were stained with DCFH-DA probe, and the intracellular ROS level was quantified by flow cytometry; d is the P value determined by CCK8 method in the presence or absence of MMP14. 216 PKF kills L02 cells; e is the CCK8 assay to determine P in the presence or absence of the MMP14 inhibitor NSC405020 216 The killing effect of PKF on 4T-1 cells.

[0064] Figure 3 This is a study on the stability and in vitro leakage of fusion protein-metal ion complexes. a is the same concentration of KLA-Fn and P under 37℃ and 60℃ conditions. 54 PKF, P 108 PKF, P 216 SDS-PAGE results of PKF after different storage time; b is the quantitative results of SDS-PAGE; c is the same concentration of KLA-Fn@Mn under 4℃ condition 2+ , P 54 PKF@Mn 2+ , P 108 PKF@Mn 2+ , P 216 PKF@Mn 2+ Leakage of Mn 2+ d is the quantification of Mn leaked from the fusion protein-metal ion complex at the same concentration under 37°C. 2+ Quantitative.

[0065] Figure 4 This is a study on the in vitro targeting of fusion proteins to tumor cells. a is P loaded with DOX 216Laser confocal scanning microscope fluorescence images of L02, MCF-7, MDA-MB-231, and 4T-1 cells after PKF treatment for 8 h; b is P loaded with DOX 216 After the cells were treated with PKF for 8 h, the intracellular fluorescence level was quantified by flow cytometry; c is the quantitative result of flow cytometry.

[0066] Figure 5 This is a study on the immune activation effect of metal ions on cells. a is the ELISA detection of IFN-β production of 4T-1 cells after stimulation with different metal ions (Na+, K+, Al3+, Zn2+, Cu2+, Ca2+, Fe2+, Fe3+, Mg2+, Mn2+) at different concentrations; b is the ELISA detection of IFN-γ production of 4T-1 cells after stimulation with different metal ions and co-incubation with PBMC; c is the flow cytometry detection of DC cell and CD8+T cell activation.

[0067] Figure 6 This is the in vivo pharmacokinetic study of the fusion protein-metal ion complex. a is the tail vein injection of P 216 a) After PKF, the serum of mice was collected to determine the content of fusion protein by ELISA; b) After tail vein injection of prototype ferritin, the serum of mice was collected to determine the content of protein by ELISA.

[0068] Figure 7 This is a study on the in vivo distribution of fusion protein-metal ion complexes. a is the tail vein injection of Cy5-ferritin, Cy5-P 216 After PKF, the Cy5 signal in the mouse body was detected and analyzed; b is the tail vein injection of Cy5-ferritin and Cy5-P 216 Detection and analysis of Cy5 signals in different tissues and organs of mice after PKF; c is the quantitative results of Cy5 signals in isolated tissues and organs.

[0069] Figure 8 The anti-tumor ability of fusion protein-metal ion complex tumor vaccine was studied. On day 0, day 5 and day 10, 3 mg / ml P 216 PKF equivalent P 216 PKF@Mn 2+ After pre-incubation with Luc-4T-1 cells for 24 hours, the "Luc-4T-1 tumor vaccine" was obtained and subcutaneously injected into the ventral side of mice. In vivo bioluminescence of Luc-4T-1 cells (a), weight changes of mice (b), tumor volume changes of mice (c) and survival curves of mice (d); e, f, g, h are subcutaneous injections of 3 mg / ml P on day 0, day 5 and day 10, respectively. 216PKF equal amounts of "Luc-GL261 tumor vaccine" were injected into the flank of mice, in vivo bioluminescence of Luc-GL261 cells (e), body weight changes of mice (f), tumor volume changes of mice (g) and survival curves of mice (h).

[0070] Fig. 9 The pharmacodynamics of fusion protein-metal ion complex in antitumor is studied. a is a schematic diagram of mouse modeling and drug administration, specifically, on days 0, 4, 8, 12, and 16, 15 mg / kg of P 216 PKF was injected into the tail vein to treat 4T-1 subcutaneous tumors. The changes in tumor volume (b), body weight (c) and survival curve (d) of mice were shown.

[0071] Fig.10 is a schematic diagram of the mechanism of action of the present invention, a is a schematic diagram of the molecular design, and b is a schematic diagram of the anti-tumor effect of P216PKF@Mn2+. DETAILED DESCRIPTION

[0072] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0073] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0074] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0075] Example 1 Preparation and characterization of fusion protein-metal ion complex prodrug:

[0076] (1) The full-length sequence of human H-ferritin was obtained using the NCBI database, and PSTAG, tumor microenvironment enzyme response sequence, and ICD induction peptide functional sequence were introduced into its N-terminus to obtain the protein sequence of the fusion protein (see Table 1 for details). After codon optimization, the cDNA of the fusion protein was constructed into the expression vector pET-28a (GenScript); then, using the prokaryotic expression system, the fusion protein-pET-28a was transformed into the expression strain BL21 (DE3) (Beyotime Biotechnology), and the expression was induced by IPTG, and then the human Fn protein was purified. The specific method is as follows: protein expression was induced by 0.5mM IPTG at 37°C for 8 hours. The bacteria were collected and resuspended in 20mM Tris-HCl (pH 8.0) buffer. The bacterial lysate was obtained by ultrasonic treatment and then centrifuged (12,000g, 30min, 4°C). The supernatant was collected and heated at 70°C for 15min, and centrifuged again (12,000g, 30min, 4°C). Collect the supernatant and use Q-Sepharose Fast Flow and superdex TM The purified protein was purified by 200 pg column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed using 12% (v / v) polyacrylamide gel. Then, the purest fraction was collected, ultrafiltered using a 100 kDa centrifugal ultrafiltration tube and the buffer was replaced with 20 mM HEPES to obtain the fusion protein.

[0077] (2) Dilute the fusion protein with 20mM HEPES pH 8.0 to a final concentration of 1.5mg / mL and a final volume of 50mL, and gently stir in a 60℃ constant temperature water bath for 30min. A manganese chloride solution (450mM) prepared with deionized water was dripped into the reaction mixture at a rate of 130μL / min (gently stirred in a 60℃ water bath) and stirred for 80min. The reaction mixture was gently stirred in a 60℃ constant temperature water bath for 160min. The reaction mixture was then cooled naturally to room temperature and centrifuged (8,000g, 30min, 4℃) to remove denatured proteins. The supernatant was placed in a 50kDa dialysis bag and dialyzed overnight with HEPES buffer at 4℃ to remove free manganese ions, thereby obtaining a fusion protein-metal ion complex.

[0078] (3) The concentration of fusion protein was determined using the BCA protein assay kit, and the encapsulated manganese was quantitatively determined using ICP-OES 5110. Dynamic light scattering (DLS) characterization of the fusion protein before and after metal loading was completed using a Zetasizer Nano ZS90 (Malvern Instruments, UK). Equal amounts of fusion protein and fusion protein-metal ion complex were measured at 25 °C.

[0079] result:

[0080] Figure 1 a is the dynamic light scattering method to characterize the fusion protein; Figure 1 c is the value of P 216 PKF@Mn 2+ Characterization of the complexes revealed that after metal loading, the fusion protein-metal ion complex had good dispersibility and uniform particle size. There was basically no difference from the fusion protein. Transmission electron microscopy (TEM) characterization of the fusion protein before and after metal loading was completed using an HT7700 microscope (Hitachi, Japan). Equal amounts of fusion protein and fusion protein-metal ion complex were measured at 25°C. Figure 1 b shows the characterization of the fusion protein by transmission electron microscopy; Figure 1 d is the value of P 216 PKF@Mn 2+ Characterization of the complex revealed that after loading with metal, the fusion protein-metal ion complex could form a 24-mer cage-shaped phase, which was basically indistinguishable from the fusion protein.

[0081] As shown in Table 2, as the length of PSTAG increases, the hydration radius of the fusion protein also increases.

[0082] KLA-Fn represents the fusion protein without PSTAG shielding sequence, which serves as a reference; P 54 PKF, P 108 PKF, P 108 PKF and P216PKF represent fusion proteins containing different PSTAG shielding sequences.

[0083] Table 1 (Sequence Listing): Description of Fusion Protein Sequences

[0084]

[0085]

[0086] The results of DLS detection of fusion protein hydration radius are shown in Table 2

[0087] Table 2: Fusion protein hydration radius

[0088]

[0089] Example 2 Study on ICD-inducing activity of fusion protein-metal ion complex

[0090] Fusion protein-metal ion complex P216PKF@Mn 2+Taking the ICD-inducing activity of the fusion protein as an example, 1 μM of the fusion protein was incubated with common human and mouse breast cancer cell lines 4T-1, MCF-7, MDA-MB-231 and mouse healthy cells L02 for 24 h, and then stained with JC-1 (green: monomer, red: aggregate) and observed for mitochondrial damage using a laser confocal scanning microscope, and a fluorescence microplate reader was used for quantitative experiments; it can be seen that L02 cells have almost no mitochondrial damage and are all red, while tumor cells have varying degrees of mitochondrial damage ( Figure 2 a, b). After staining with DCFH-DA probe, intracellular ROS levels were quantified by flow cytometry.

[0091] result:

[0092] Fusion protein-metal ion complex increases ROS levels in tumor cells ( Figure 2 c). 4T-1 cells were incubated with 1 μM NSC405020 for 6 h, then incubated with fresh medium containing PPKF (0-20 μM, 2-fold gradient dilution) for 24 h, and then 10 μl of CCK8 detection reagent was added to each well and incubated at 37°C for 1.5 h. The absorbance (OD) at 450 nm was measured using a Spectra Max I3X microplate reader. 216 The killing effect of PKF on 4T-1 cells was inhibited by the MMP14 inhibitor NSC405020 ( Figure 2 d). After adding 100ug / mL MMP14 enzyme to L02 cells and incubating for 24h, 10uL CCK-8 solution was added to each well and incubated for 1.5h, and the absorbance (OD) at 450nm was measured. The results showed that in the presence of MMP14 enzyme, P 216 PKF can produce MMP14-dependent cytotoxicity ( Figure 2 e). This indicates that the fusion protein-metal ion complex can successfully induce ICD in tumor cells in a MMP14-dependent manner, while causing little damage to healthy cells.

[0093] Example 3 Stability and in vitro leakage study of fusion protein-metal ion complex before and after metal loading

[0094] 1) To study the stability of the fusion protein, 1 mL of the fusion protein (2 mg / mL) was placed under stable conditions at 37°C or 60°C. At the desired time points (0, 2, 4, 8, 24, 72 h), 10 μL of sample was taken and quantified by SDS-PAGE.

[0095] result:

[0096] Figure 3a, b are the SDS-PAGE results of the fusion protein after being stored for different time periods. It was found that the fusion protein was very stable at 37°C and 60°C, with very little degradation.

[0097] 2) To study the in vitro leakage of the fusion protein-metal ion complex, 1 mL of the fusion protein-metal ion complex (0.75 mg / mL based on the amount of fusion protein) was placed in a 50 kDa dialysis bag, and then placed in a container containing 5 mL of the same buffer and stirred continuously at 4°C or 37°C. At the desired time points (0, 1, 2, 4, 6, 8, 12, 24 h), 200 μL of the dialysate was drawn and the manganese was quantified by the formaldehyde oxime photometry. The results were expressed as the percentage of release.

[0098] result:

[0099] Figure 3 c and d are the leakage results of the fusion protein-metal ion complex after being stored for different time periods. It was found that the leakage of the fusion protein-metal ion complex within 24 hours was less than 10% at 4°C and 37°C, and the leakage was very small.

[0100] Example 4 Study on the targeting of fusion protein-metal ion complex to tumor cells in vitro

[0101] In order to study whether the fusion protein-metal ion complex prodrug system has targeting effect on tumor cells in vitro, common human and mouse breast cancer cells 4T-1, MCF-7, MDA-MB-231 and human healthy liver cells L02 were selected for research. After incubation of several cells with the fusion protein-doxorubicin complex encapsulated with doxorubicin for 8 hours in vitro, the cell flow cytometry and laser confocal microscopy methods were used to detect the entry of the fusion protein-doxorubicin complex into tumor cells.

[0102] result:

[0103] like Figure 4 It was shown that the fusion protein-doxorubicin complex could preferentially enter tumor cells rather than healthy cells and had good tumor selectivity.

[0104] Example 5 Study on the immune activation effect of metal ions on cells

[0105] As an important metal ion in tumor immunotherapy, Mg 2+ , Mn 2+ 、Al 3+ 、Zn 2+Etc. have shown cytokine-like immunomodulatory functions. In order to screen out metal ions with immune response activity that activates mature DCs and activated CD8+T cells through the STING pathway. 4T-1 cells were incubated with different concentrations of metal ions and then co-incubated with PBMCs to detect cytokine secretion and immune cell activation.

[0106] result

[0107] like Figure 5 Display, Mn 2+ The production of IFN-β in tumor cells was significantly increased in a dose-dependent manner ( Figure 5 a) Mn 2+ After the treated tumor cells were co-cultured with PBMC, the IFN-γ level in the co-culture supernatant was also significantly increased ( Figure 5 b). Flow cytometry results showed that Na + 、Al 3+ 、Zn 2+ , Cu 2+ , K + , Ca 2+ , Fe 2+ , Fe 3+ Mg 2+ , Mn 2+ etc. can increase DC cells and CD8 + T cell activation ( Figure 5 c) These results indicate that the above metal ions can induce better immune response activation.

[0108] Example 6 In vivo pharmacokinetic study of fusion protein-metal ion complex

[0109] To study the pharmacokinetic properties of the fusion protein-metal ion complex in vivo and compare it with the prototype ferritin, we injected equal amounts of the prototype ferritin and the fusion protein-metal ion complex (5 mg / kg mouse body weight) into female BALB / c mice (3 mice per group) via the tail vein. Blood was collected from the retro-orbital venous plexus at 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h after injection. The supernatant was collected after centrifugation at 1000 g for 5 min. The concentration of serum ferritin was determined using a ferritin detection ELISA kit (Genscript). The plasma half-life was calculated using GraphPadPrism 9.0.

[0110] result:

[0111] like Figure 6 The results showed that the blood half-life of the fusion protein-metal ion complex increased significantly, indicating that the fusion protein-metal ion complex has a longer drug retention time in the systemic circulation. This also allows the drug to continue to accumulate at the tumor site.

[0112] Example 7 Study on the in vivo distribution of fusion protein-metal ion complex

[0113] In order to further explore whether the fusion protein-metal ion complex can specifically accumulate at the tumor site in vivo, we conducted a study on the in vivo distribution of the fusion protein-metal ion complex. In vivo, the Cy5-NHS-labeled fusion protein-metal ion complex was injected into the tail vein of tumor-bearing mice. The in vivo imaging system was used to further study the in vivo distribution of the Cy5 signal over time and whether it was targeted to the 4T-1 transplanted tumor. The Cy5 signal was detected and quantified in the mouse ex vivo tissue.

[0114] result:

[0115] like Figure 7 The results showed that the Cy5.5-NHS-labeled fusion protein-metal ion complex can reach the tumor site and be enriched in about 8 hours, which has better tumor targeting than the prototype ferritin control without environmental response. In addition, the fusion protein-metal ion complex is less enriched in the liver, which improves the safety of the system.

[0116] Example 8 Prevention of Tumor Formation by Fusion Protein-Metal Ion Complex

[0117] As an ICD-based anti-tumor platform, the most direct test effect is to see the prevention of tumor formation. 216 PKF equivalent P 216 PKF@Mn 2+ After pre-incubation with Luc-4T-1 cells for 24 hours, the "Luc-4T-1 tumor vaccine" was obtained and subcutaneously injected into the ventral side of mice. The in vivo bioluminescence of the Luc-4T-1 cells, the weight changes of the mice, the changes in the tumor volume of the mice and the survival curve of the mice were observed and recorded. Similarly, the "Luc-GL261 tumor vaccine" was prepared and inoculated into the ventral side of mice. The in vivo bioluminescence of the Luc-GL261 cells, the weight changes of the mice, the changes in the tumor volume of the mice and the survival curve of the mice were observed and recorded.

[0118] result:

[0119] like Figure 8 It was shown that the ICD-based fusion protein-metal ion complex tumor vaccine had a strong inhibitory effect on the growth of subcutaneous tumors of the same cancer species and effectively inhibited tumor recurrence.

[0120] Example 9: Anti-tumor pharmacodynamics study of fusion protein-metal ion complex

[0121] To test the killing and inhibitory effects of the fusion protein-metal ion complex prodrug system on tumors, antitumor pharmacodynamics studies were conducted. Balb / c mice were subcutaneously inoculated with 1×10 6 4T-1 cells were used to construct a breast cancer subcutaneous tumor model. On days 0, 4, 8, 12, and 16, 15 mg / kg P 216 PKF was injected into the tail vein in equal amounts to treat 4T-1 subcutaneous tumors, and the changes in tumor volume, weight and survival curves of mice were observed and recorded.

[0122] result:

[0123] like Fig. 9 The results showed that the fusion protein-metal ion complex significantly inhibited tumor growth in Balb / c mice bearing 4T-1 cells.

Claims

1. A tumor microenvironment-responsive fusion protein-metal ion complex prodrug system, characterized in that: The system includes a tumor-targeted ICD-inducing drug carrier and an immune-activating metal ion content.

2. The system according to claim 1, characterized in that The tumor-targeted ICD-inducing drug carrier is a human heavy chain ferritin derivative that is polymerized to form nanoparticles with cavities, and the metal ions are loaded in the cavities.

3. The system according to claim 2, characterized in that The human heavy chain ferritin derivative spontaneously forms a cage-like 24-mer that encapsulates metal ions.

4. The system according to claim 3, characterized in that The human heavy chain ferritin derivative comprises a PSTAG shielding sequence, a tumor microenvironment enzyme response sequence, an ICD inducing peptide, an intracellular release sequence and an intraluminal modified human heavy chain ferritin; the metal ions comprise manganese, magnesium, copper, zinc, iron, aluminum, potassium or sodium.

5. The system according to claim 4, characterized in that The PSTAG shielding sequence is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; The tumor microenvironment enzyme response sequence is SEQ ID NO.4; The ICD inducing peptide is SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12; The intracellular release sequence is SEQ ID NO.13; The amino acid sequence of the lumen-modified human heavy chain ferritin is SEQ ID NO.

14.

6. The system according to claim 5, characterized in that The amino acid sequence of the human heavy chain ferritin derivative is SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 or SEQ ID NO.

18.

7. The method for preparing the system according to claim 6, characterized in that: The method comprises the following steps: fusing and expressing an ICD inducing peptide on the outside of ferritin; utilizing the inherent ion channel of the ferritin structure, opening the ferritin ion channel and the PSTAG winding shield by heating, adding metal ions thereto to allow them to enter the ferritin, restoring the temperature to room temperature so that the ferritin ion channel is closed and the PSTAG restores the spatial shielding, thereby obtaining a fusion protein-metal ion complex whose inner cavity contains metal ions.

8. Use of the system according to any one of claims 1 to 6 in preparing drugs for preventing or treating tumors.

9. The use according to claim 8, characterized in that: The tumor is breast cancer, colorectal cancer, lung cancer, ovarian cancer, melanoma, gastric cancer, pancreatic cancer, bladder cancer, kidney cancer, prostate cancer or brain cancer.

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