A tumor microenvironment-responsive fusion protein-metal ion complex prodrug system, and a preparation method and application thereof

Abstract

The application provides a novel platform which can be used for tumor immunotherapy. The platform is self-assembled from full-length human ferritin which is specifically targeted to tumor microenvironment and induces ICD death of tumors, and is jointly expressed by a protease-activatable poly-peptide prodrug mask and a mitochondria-destroying peptide. Meanwhile, the platform has ion channels on the protein shell, and can load immune-active metal ions in the cavity thereof, so as to synergistically activate the spatial and temporal anti-tumor immunity when ICD occurs. Since the mitochondria-destroying peptide and the metal ions can be optimized according to the tumor type, the platform can be used for treating various tumors.

Description

Technical Field

[0001] This invention relates to a fusion protein-metal ion complex prodrug system that responds to the tumor microenvironment, its preparation method, and its application. Background Technology

[0002] Immunogenic cell death (ICD) is a mechanism that generates an anti-tumor immune response. ICD promotes the expression of calreticulin (CRT), acting as a "eat me" signal, and releases damage-associated molecular patterns (DAMPs) and tumor-associated antigens, which can serve as in situ tumor vaccines. Most conventional anticancer drugs used to induce ICD achieve this by triggering "side branch effects," resulting in a limited anti-tumor immune response. For effective ICD induction, the generation of persistent reactive oxygen species (ROS) leading to endoplasmic reticulum (ER) stress is crucial. Given that mitochondria are the primary organelles for ROS production in eukaryotic cells, directly manipulating mitochondria to enhance ROS production may be a more direct and effective approach. Bioactive peptides, such as naturally occurring toxic peptides, borane mimics, and cationic amphiphilic peptides, can induce mitochondrial membrane rupture, leading to ROS accumulation 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] Meanwhile, achieving spatiotemporal synchronization of tumor-specific immune responses after stereotactic induction by ICDs also presents challenges. Metal ions, such as aluminum, magnesium, zinc, and manganese, are increasingly recognized for their crucial roles in regulating innate immunity. Therefore, metal ions may be considered potential enhancers of tumor-specific immune responses. However, highly water-soluble metal ions are difficult to load correctly into conventional delivery systems, leading to low loading efficiency and premature leakage during circulation. Furthermore, under physiological conditions, PO... 3- The abundant presence of metal ions can lead to the formation of phosphate nanoparticles upon leakage, resulting in 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 distributed iron storage protein composed of 24 subunits that aggregate to form a cage-like structure with an outer diameter of 12 nm and an inner lumen of 8 nm. This cage-like structure can encapsulate metal ions such as manganese, magnesium, copper, zinc, iron, aluminum, potassium, and sodium. More importantly, CD71, as the receptor for Fn, is overexpressed in most human tumors, allowing Fn to be preferentially internalized by tumor cells. Due to its reversible cage-like structure and natural metal encapsulation properties, as well as its thermal stability, biocompatibility, and non-immunogenicity, Fn has become an ideal tumor-targeting delivery platform for metal-based therapies. However, Fn still faces several challenges. First, CD71 is significantly expressed in the liver, leading to preferential hepatic interception of Fn rather than targeting the tumor. Second, the stability of Fn-drug complexes is not ideal, with over 30% drug leakage observed within the first week. Furthermore, Fn has a relatively short plasma half-life (approximately 2 hours).

[0005] Therefore, there is a need in the art for formulations or complexes that can overcome the defects of Fn and simultaneously achieve spatiotemporal synchronous activation of tumor-specific immune responses after stereotactic induction by ICD. Summary of the Invention

[0006] Purpose of the invention

[0007] The purpose of this invention is to provide a tumor microenvironment-responsive fusion protein-metal ion complex prodrug system, overcoming the shortcomings of existing technologies.

[0008] Technical solution

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

[0010] The system is characterized in that the tumor-targeting ICD-induced drug carrier is polymerized from human heavy chain ferritin derivatives to form cavitary nanoparticles, and the metal ions are loaded within 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-induced peptide, an intracellular release sequence, and a lumen-modified human heavy chain ferritin; the metal ions comprise manganese, magnesium, copper, zinc, iron, aluminum, potassium, or sodium.

[0013] The PSTAG masking sequence is as follows:

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

[0015] or

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

[0017] or

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

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

[0020] The ICD-inducible peptides are GQVGRQLAIIGDDINR (see SEQ ID NO. 5); CGTNVFNATFHIWHSGQFGT (see SEQ ID NO. 6); and 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 as follows:

[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 QINLELYASYVYLSMSYYFDDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNESHHHHHHHH, see SEQ ID NO.18.

[0037] The method for preparing the system is characterized by comprising the following steps: fusing an ICD-inducible peptide to the outside of ferritin; utilizing the ion channels inherent in the ferritin structure, heating is used to open the ferritin ion channels and PSTAG entanglement shield, metal ions are added to them to allow them to enter the ferritin, and the ferritin ion channels are closed and the PSTAG spatial shield is restored at room temperature, resulting in a fusion protein-metal ion complex containing metal ions in its lumen.

[0038] The application of the system in the preparation of drugs for the prevention or treatment of 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-like 24-mer.

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

[0041] This invention also relates to a method for preparing the aforementioned fusion protein-metal ion complex prodrug system, specifically, the method includes the following steps:

[0042] Step (1): Prepare and purify the lumen-modified human heavy chain ferritin derivative.

[0043] The full-length sequence of human H-ferritin was obtained from the NCBI database. PSTAG, a tumor microenvironment response site, and an ICD-induced peptide functional sequence were introduced at 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, using a prokaryotic expression system, the fusion protein pET-28a was transformed into the expression strain BL21(DE3) (Beyotime Biotechnology), and expression was induced by IPTG. The human HFn protein was then purified.

[0044] The specific methods for expression and purification are as follows: Protein expression was induced at 37℃ for 8 hours with 0.5 mM IPTG. Bacterial cells were collected and resuspended in 20 mM Tris-HCl (pH 8.0) buffer. Bacterial lysates were obtained by sonication and then centrifuged (12,000 g, 30 min, 4℃). The supernatant was collected and heated at 70℃ for 15 min, then centrifuged again (12,000 g, 30 min, 4℃). The supernatant was then purified using Q-Sepharose FastFlow and Superdex. TM Purification was performed using a 200 pg column. Analysis was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% (v / v) polyacrylamide gel. The purest fraction was then collected, ultrafiltered using a 100 kDa centrifugal ultrafiltration tube, and the buffer was replaced with 20 mM HEPES to obtain the fusion protein concentrate.

[0045] Step (2): Load manganese ions using lumen-modified human heavy chain ferritin derivatives.

[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 and a final volume of 50 mL, and then gently stirred in a 60 °C water bath for 30 min.

[0047] A manganese chloride solution (450 mM) prepared with deionized water was pumped dropwise into the reaction mixture at a rate of 130 μL / min (while gently stirring in a water bath at 60 °C) 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 allowed to cool naturally to room temperature 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 overnight at 4°C with HEPES buffer to remove free manganese ions, thus obtaining the fusion protein-metal ion complex.

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

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

[0052] principle:

[0053] like Figure 10 As shown in Figure a, the human heavy chain ferritin derivative is a fusion protein formed by coupling a PSTAG shielding sequence, a tumor microenvironment enzyme response sequence, an ICD-inducible sequence, and an intracellular release sequence with the lumen-modified human heavy chain ferritin. It achieves the following functions: ① PSTAG shielding sequence: reduces leakage of metal ions within the ferritin cage, prolongs the half-life of the fusion protein, improves its stability, enhances its in vivo distribution, reduces off-target liver accumulation, and lowers off-target toxicity. ② Tumor microenvironment response site: can be cleaved by enzymes highly expressed in the tumor microenvironment, removing the shielding sequence and releasing the fusion protein to exert anti-tumor effects. ③ ICD-inducible peptide: a cytotoxic peptide that can act on tumor cell mitochondria and induce ICD-induced cell death. ④ Intracellular cleavage site: after ferritin carries the ICD-inducible short peptide into tumor cells, it can separate the ICD-inducible short peptide from ferritin, allowing the ICD-inducible short peptide to act on mitochondria and exert its ICD-inducing effect.

[0054] like Figure 10 As shown in b, the fusion protein-metal ion complex prodrug system exhibits strong antitumor activity after intravenous injection. Its mechanism of action is as follows: after entering the body, the complex undergoes MMP14 cleavage in the tumor microenvironment, removing PSTAG and exposing KLA-Fn, which then enters cells via CD71-mediated entry. The active molecule KLA is released after intracellular cleavage, acting on mitochondria and inducing an increase in ROS levels. Tumor cells then undergo ICD and STING pathway activation, thereby mediating DC maturation and T cell activation, achieving a systemic immune response. In this invention, experimental results show that the fusion protein-metal ion complex can successfully induce ICD in tumor cells and has good tumor targeting, effectively inhibiting tumor development and progression while activating immune cell activity.

[0055] Beneficial effects

[0056] This invention constructs a novel fusion protein-metal ion complex prodrug system, comprising a tumor-targeting ICD-induced drug carrier and an immune-activating metal ion content. The tumor-targeting ICD-induced drug carrier is formed by polymerizing a human heavy chain ferritin derivative to create a hollow nanoparticle, within which the metal ion and drug are loaded. This invention has the following advantages:

[0057] 1. For example Figure 7 As shown, this complex significantly reduces liver accumulation, overcoming the limitation of existing technologies where Fn is preferentially intercepted by the liver rather than targeting tumors; Figure 6 The extended half-life is shown, as... Figure 7 As shown, it reached tumor site enrichment in approximately 8 hours, exhibiting better tumor targeting compared to the unresponsive proto-ferritin control. Furthermore, the fusion protein-metal ion complex showed less accumulation in the liver, improving system safety.

[0058] 2. For example Figure 3 As shown, the leakage of this complex was less than 10% within 24 hours under conditions of 4℃ and 37℃, which is very low and overcomes the technical defect of unsatisfactory stability of Fn-drug complexes in the prior art.

[0059] 3. For example Figure 2 As shown, the complex can successfully induce ICD in tumor cells in an MMP14-dependent manner, while causing minimal damage to healthy cells, indicating that the present invention has less toxicity and is safer.

[0060] 4. For example 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.) Increases the activation of dendritic cells (DCs) and CD8+ T cells, inducing better immune response activation. For example... Figure 8 As shown, the complex of the present invention can itself be used as a drug to prevent the occurrence of tumors. For example... Figure 9 As shown, it can also inhibit tumor growth.

[0061] 5. For example Figure 4 As shown, the present invention can further be used as a carrier to encapsulate drugs (anti-tumor drugs, such as doxorubicin) and increase their anti-tumor effects. Attached Figure Description

[0062] Figure 1 Characterization of a series of luminal modified human heavy chain ferritin derivatives and fusion protein-metal ion complexes. a represents the effect of KLA-Fn and P under the same protein concentration conditions. 54 PKF, P 108 PKF, P 216Dynamic light scattering results of PKF; b shows the results of KLA-Fn and P using transmission electron microscopy. 54 PKF, P 108 PKF, P 216 Characterized by PKF; c is P 216 PKF@Mn 2+ Dynamic light scattering results of the complex; d represents P 216 PKF@Mn 2+ Transmission electron microscopy characterization of the complex.

[0063] Figure 2 This study investigates the ICD-induced activity of the fusion protein in vitro. a represents 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 represents P 216 Quantitative results of cells treated with PKF and stained with JC-1 using a fluorescent microplate reader; c represents P 216 Cells treated with PKF were stained with the DCFH-DA probe, and intracellular ROS levels were quantified by flow cytometry; d represents the P content measured using the CCK8 assay in the presence or absence of MMP14. 216 The killing effect of PKF on L02 cells; e represents the P-value measured by the CCK8 assay in the presence or absence of the MMP14 inhibitor NSC405020. 216 The killing effect of PKF on 4T-1 cells.

[0064] Figure 3 This study investigates the stability and in vitro leakage of the fusion protein-metal ion complex. (a) represents the same concentrations of KLA-Fn and P at 37℃ and 60℃. 54 PKF, P 108 PKF, P 216 SDS-PAGE results of PKF after different storage times; b is the quantitative result of SDS-PAGE; c is the result of KLA-Fn@Mn at the same concentration under 4℃ conditions. 2+ P 54 PKF@Mn 2+ P 108 PKF@Mn 2+ P 216 PKF@Mn 2+ Leaking Mn 2+ Quantitative analysis; d represents the amount of Mn leaked from the same concentration of the fusion protein-metal ion complex at 37℃. 2+ Quantitatively.

[0065] Figure 4 This study investigates the in vitro targeting of the fusion protein to tumor cells. 'a' represents P loaded with DOX. 216Laser confocal scanning microscopy fluorescence images of L02, MCF-7, MDA-MB-231, and 4T-1 cells after 8 hours of PKF treatment; b is a P-type cell loaded with DOX. 216 After PKF treatment for 8 hours, intracellular fluorescence levels were quantified by flow cytometry; c represents the quantitative results of flow cytometry.

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

[0067] Figure 6 This study investigates the in vivo pharmacokinetics of the fusion protein-metal ion complex. (a) shows the effect of tail vein injection of P. 216 After PKF, mouse serum was collected and the content of fusion protein was determined by ELISA; b was mouse serum collected and the protein content was determined by ELISA after tail vein injection of proto-ferritin.

[0068] Figure 7 This study investigates the in vivo distribution of the fusion protein-metal ion complex. a) Intravenous injection of Cy5-ferritin and Cy5-P... 216 Following PKF, Cy5 signaling in mice was detected and analyzed; b represents tail vein injection of Cy5-ferritin and Cy5-P. 216 Cy5 signal detection and analysis in different tissues and organs of mice after PKF; c represents the quantitative results of Cy5 signal in isolated tissues and organs.

[0069] Figure 8 This study investigated the antitumor activity of a fusion protein-metal ion complex tumor vaccine. On days 0, 5, and 10, the vaccine was administered at a dose of 3 mg / ml of P... 216 PKF equal amount of P 216 PKF@Mn 2+ After pre-incubation with Luc-4T-1 cells for 24 hours, a "Luc-4T-1 tumor vaccine" was obtained. This vaccine was subcutaneously injected into the ventral side of mice. In vivo bioluminescence of Luc-4T-1 cells was observed (a), mouse weight changes (b), tumor volume changes (c), and mouse survival curves (d). E, F, G, and H represent subcutaneous injections of 3 mg / ml P on days 0, 5, and 10, respectively. 216An equal amount of PKF was administered to the ventral side of mice as “Luc-GL261 tumor vaccine”. In vivo bioluminescence of Luc-GL261 cells (e), changes in mouse body weight (f), changes in tumor volume in mice (g), and survival curves of mice (h) were observed.

[0070] Figure 9 This study investigates the antitumor pharmacodynamics of the fusion protein-metal ion complex. Figure a shows a schematic diagram of mouse modeling and drug administration, specifically, on days 0, 4, 8, 12, and 16, P was administered at a dose of 15 mg / kg. 216 PKF was administered via tail vein injection to treat 4T-1 subcutaneous tumors in mice. Tumor volume changes (b), body weight changes (c), and survival curves (d) were observed in mice.

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

[0072] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0073] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0074] In the embodiments 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 embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

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

[0076] (1) The full-length sequence of human H-ferritin was obtained from the NCBI database. PSTAG, tumor microenvironment enzyme response sequences, and ICD-induced peptide functional sequences were introduced at its N-terminus to obtain the protein sequence of the fusion protein (see Table 1). After codon optimization, the cDNA of the fusion protein was constructed into the expression vector pET-28a (GenScript). Then, using a prokaryotic expression system, the fusion protein pET-28a was transformed into the expression strain BL21(DE3) (Beyotime Biotechnology), and expression was induced by IPTG. The human Fn protein was then purified. Specifically, the protein was induced to express at 37°C with 0.5 mM IPTG for 8 hours. The bacterial cells were collected and resuspended in 20 mM Tris-HCl (pH 8.0) buffer. Bacterial lysate was obtained by sonication and then centrifuged (12,000 g, 30 min, 4°C). The supernatant was collected and heated at 70°C for 15 min, then centrifuged again (12,000 g, 30 min, 4°C). Supernatant was collected using Q-Sepharose Fast Flow and superdex. TM Purification was performed using a 200 pg column. Analysis was then performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% (v / v) polyacrylamide gel. The purest fraction was then 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) The fusion protein was diluted with 20 mM HEPES pH 8.0 to a final concentration of 1.5 mg / mL, with a final volume of 50 mL, and gently stirred in a 60°C water bath for 30 min. A 450 mM manganese chloride solution prepared with deionized water was added dropwise to the reaction mixture at a rate of 130 μL / min (while gently stirring in a 60°C water bath), and stirred for 80 min. The reaction mixture was then gently stirred in a 60°C water bath for 160 min. The reaction mixture was then allowed to cool naturally to room temperature and centrifuged (8,000 g, 30 min, 4°C) to remove denatured protein. The supernatant was placed in a 50 kDa dialysis bag and dialyzed overnight with HEPES buffer at 4°C to remove free manganese ions, thus obtaining the fusion protein-metal ion complex.

[0078] (3) The concentration of the fusion protein was determined using a BCA protein assay kit, and the encapsulated manganese was quantitatively determined using an ICP-OES 5110. Dynamic light scattering (DLS) characterization of the fusion protein before and after metal loading was performed using a Zetasizer NanoZS90 (Malvern Instruments, UK). Equal amounts of the fusion protein and the fusion protein-metal ion complex were measured at 25°C.

[0079] result:

[0080] Figure 1 a) The dynamic light scattering method is used to characterize the fusion protein; Figure 1 c represents P 216 PKF@Mn 2+ Characterization of the complex revealed that the fusion protein-metal ion complex exhibited good dispersibility and uniform particle size after metal loading, showing virtually no difference from the fusion protein itself. Transmission electron microscopy (TEM) characterization of the fusion protein before and after metal loading was performed using an HT7700 microscope (Hitachi, Japan). Equal amounts of the fusion protein and the fusion protein-metal ion complex were measured at 25°C. Figure 1 b shows the characterization of the fusion protein using transmission electron microscopy; Figure 1 d represents the pair 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-like conformation, essentially indistinguishable from the fusion protein itself.

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

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

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

[0084]

[0085]

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

[0087] Table 2: Hydration radius of fusion proteins

[0088]

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

[0090] The fusion protein-metal ion complex P216PKF@Mn 2+To study the ICD-induced activity of the fusion protein, 1 μM of the fusion protein was incubated with common human and mouse breast cancer cells 4T-1, MCF-7, MDA-MB-231, and healthy mouse L02 cells for 24 h. After staining with JC-1 (green: monomers, red: aggregates), mitochondrial damage was observed using a laser confocal scanning microscope, and quantitative experiments were performed using a fluorescent microplate reader. It was observed that L02 cells showed almost no mitochondrial damage, appearing entirely red, while tumor cells showed varying degrees of mitochondrial damage. Figure 2 a, b). After staining with the DCFH-DA probe, intracellular ROS levels were quantified by flow cytometry.

[0091] result:

[0092] The fusion protein-metal ion complex increased the ROS level in tumor cells. Figure 2 c). After incubating 4T-1 cells with 1 μM NSC405020 for 6 h, they were incubated with fresh medium containing PPKF (0-20 μM, 2-fold serial dilutions) for 24 h. Then, 10 μL of CCK8 assay 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 is inhibited by the MMP14 inhibitor NSC405020. Figure 2 d). In L02 cells, after incubation with 100 μg / mL MMP14 enzyme for 24 h, 10 μL of CCK-8 solution was added to each well and incubated for 1.5 h. The absorbance (OD) at 450 nm was then measured. The results showed that in the presence of MMP14 enzyme, P 216 PKF can produce MMP14-dependent cytotoxicity ( Figure 2 e). This demonstrates that the fusion protein-metal ion complex can successfully induce ICD in tumor cells in a MMP14-dependent manner, while causing minimal damage to healthy cells.

[0093] Example 3: Stability and in vitro leakage of the 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 incubated at 37 °C or 60 °C under stable conditions. 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 and b show the SDS-PAGE results of the fusion protein after storage for different times. The fusion protein was found to be very stable at both 37°C and 60°C, with minimal degradation.

[0097] 2) To investigate 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, which was then placed in a container containing 5 mL of the same buffer solution. The mixture was continuously stirred at 4°C or 37°C. At the desired time points (0, 1, 2, 4, 6, 8, 12, and 24 h), 200 μL of the dialysate was aspirated, and manganese was quantified using the formaldehyde oxime spectrophotometric method. The results were expressed as a percentage release.

[0098] result:

[0099] Figure 3 c and d show the leakage results of the fusion protein-metal ion complex after storage for different times. It was found that the leakage of the fusion protein-metal ion complex within 24 hours was less than 10% under conditions of 4℃ and 37℃, which is very low.

[0100] Example 4: In vitro targeting study of tumor cells by the fusion protein-metal ion complex

[0101] To investigate the targeting ability of a fusion protein-metal ion complex prodrug system on tumor cells in vitro, common human and mouse breast cancer cells 4T-1, MCF-7, and MDA-MB-231, as well as healthy human hepatocytes L02, were selected for the study. In vitro, after incubating the various cell types with a doxorubicin-encapsulated fusion protein-doxorubicin complex for 8 hours, flow cytometry and laser confocal microscopy were used to detect the entry of the fusion protein-doxorubicin complex into the tumor cells.

[0102] result:

[0103] like Figure 4 The results showed that the fusion protein-doxorubicin complex could preferentially enter tumor cells rather than healthy cells, exhibiting excellent 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, such as Mg 2+ Mn 2+ Al 3+ Zn 2+Cells have already demonstrated cytokine-like immunomodulatory functions. To screen for metal ions that activate immune responses in mature dendritic cells (DCs) and activated CD8+ T cells via the STING pathway, different concentrations of metal ions were incubated with 4T-1 cells and then co-incubated with PBMCs. Cytokine secretion and immune cell activation were then assessed.

[0106] result

[0107] like Figure 5 The display shows that Mn 2+ It significantly increased the production of IFN-β in tumor cells in a dose-dependent manner. Figure 5 a). Mn 2+ After co-culturing the treated tumor cells with PBMCs, the IFN-γ level in the supernatant of the co-culture 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+ These can all increase DC cells and CD8. + T cell activation ( Figure 5 c). These results indicate that the aforementioned metal ions can all induce better immune response activation.

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

[0109] To investigate the pharmacokinetics of the fusion protein-metal ion complex in vivo and compare it with that of proto-ferritin, equal amounts of proto-ferritin and the fusion protein-metal ion complex (5 mg / kg mouse body weight) were injected into female BALB / c mice (n=3 per group) via tail vein injection. Blood samples were collected from the retroorbital venous plexus at 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h post-injection. The supernatant was collected after centrifugation at 1000 g for 5 min. Serum ferritin concentration was determined using a ferritin detection ELISA kit (Genscript). Plasma half-life was calculated using GraphPadPrism 9.0.

[0110] result:

[0111] like Figure 6 The study showed a significant increase in the blood half-life of the fusion protein-metal ion complex, indicating a longer drug retention time in the systemic circulation. This allows the drug to accumulate continuously at the tumor site.

[0112] Example 7: In vivo distribution study of the fusion protein-metal ion complex

[0113] To further investigate whether the fusion protein-metal ion complex can specifically accumulate at tumor sites in vivo, we conducted an in vivo distribution study of the fusion protein-metal ion complex. In vivo, tumor-bearing mice were administered the Cy5-NHS-labeled fusion protein-metal ion complex via tail vein injection. In vivo imaging was used to further investigate the in vivo distribution of Cy5 signal over time and whether it targets 4T-1 xenografts. Cy5 signal was detected and quantified in mouse tissue samples.

[0114] result:

[0115] like Figure 7 The results showed that the Cy5.5-NHS-labeled fusion protein-metal ion complex could reach tumor site enrichment in about 8 hours, exhibiting better tumor targeting compared to the unresponsive proto-ferritin control. Furthermore, the fusion protein-metal ion complex showed less accumulation in the liver, improving system safety.

[0116] Example 8: Prevention of tumor formation by fusion protein-metal ion complex

[0117] As an ICD-based anti-tumor platform, the most direct way to test its effectiveness is to examine its preventive effect against tumor formation. On days 0, 5, and 10, P was administered at a dose of 3 mg / ml. 216 PKF equal amount of P 216 PKF@Mn 2+ After pre-incubating Luc-4T-1 cells for 24 hours, a "Luc-4T-1 tumor vaccine" was obtained. This vaccine was subcutaneously injected into the ventral side of mice, and the in vivo bioluminescence of Luc-4T-1 cells, changes in mouse body weight, changes in tumor volume, and survival curves were observed and recorded. Similarly, a "Luc-GL261 tumor vaccine" was prepared and inoculated into the ventral side of mice, and the in vivo bioluminescence of Luc-GL261 cells, changes in mouse body weight, changes in tumor volume, and survival curves were observed and recorded.

[0118] result:

[0119] like Figure 8 The results showed 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 type and effectively inhibited tumor recurrence.

[0120] Example 9: Pharmacodynamic study of the antitumor activity of the fusion protein-metal ion complex.

[0121] To examine the tumor-killing and inhibitory effects of the fusion protein-metal ion complex prodrug system, an antitumor pharmacodynamic study was conducted. Balb / c mice were subcutaneously inoculated with 1×10⁻⁶ mol / L mol / L. 6 A subcutaneous breast cancer tumor model was constructed using 4T-1 cells. On days 0, 4, 8, 12, and 16, P was administered at a dose of 15 mg / kg. 216 PKF was administered via tail vein injection to treat 4T-1 subcutaneous tumors in mice. Changes in tumor volume, body weight, and survival curves were observed and recorded.

[0122] result:

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

Claims

1. A tumor microenvironment-responsive fusion protein-metal ion complex prodrug system, characterized in that, The system includes a tumor-targeting ICD-induced drug carrier and immune-activating metal ion contents; The tumor-targeting ICD-induced drug carrier is polymerized from human heavy chain ferritin derivatives to form cavitary nanoparticles, and the metal ions are loaded within the cavities. The human heavy chain ferritin derivative spontaneously forms a cage-like 24-mer that encapsulates metal ions. The human heavy chain ferritin derivative comprises a PSTAG shielding sequence, a tumor microenvironment enzyme response sequence, an ICD-induced peptide, an intracellular release sequence, and luminal-modified human heavy chain ferritin; the metal ion is Na. + K + Al 3+ Zn 2+ Cu 2+ Ca 2+ Fe 2+ Fe 3+ Mg 2+ or Mn 2+ ; The amino acid sequence of the human heavy chain ferritin derivative is SEQ ID NO.16, SEQ ID NO.17 or SEQ ID NO.

18.

2. The method for preparing the system according to claim 1, characterized in that, The process includes the following steps: fusion expression of ICD-induced peptides on the outside of ferritin; utilizing the ion channels inherent in the ferritin structure, heating is used to open the ferritin ion channels and PSTAG entanglement shield, metal ions are added to allow them to enter the ferritin, and the ferritin ion channels are closed and the PSTAG spatial shield is restored at room temperature, resulting in a fusion protein-metal ion complex containing metal ions in the lumen.

3. The use of the system according to any one of claims 2 in the preparation of a drug for treating breast cancer.

Images